Biochemistry: A Practical Manual: March 2019

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Editors
Suresh Kumar, Shelly Praveen and Aruna Tyagi
DIVISION OF BIOCHEMISTRY
ICAR-Indian Agricultural Research Institute
New Delhi-110012
Citation:
Suresh Kumar, Shelly Praveen and Aruna Tyagi (2019) Biochemistry: A
Practical Manual. Division of Biochemistry, ICAR-Indian Agricultural
Research Institute, New Delhi-110012.
© The Editors
Printed :
March 2019
Published by:
Post Graduate School,
ICAR-Indian Agricultural Research Institute
New Delhi-110012
Editors: Suresh Kumar, Shelly Praveen and Aruna Tyagi
Chapter Page
Title of the Chapter Authors
No. No.
Measurement of pH and preparation of Dr. Suresh Kumar
1. 1
reagents, standard acids, alkali and buffers.
Estimation carbohydrates. Dr. Vinutha T. and Veda
2. 7
Krishnan
Extraction of oil and estimation of its Dr. Vinutha T. and Navita
3. 20
characteristic features. Bansal
4. Estimation of Iodine value of an oil sample Vinutha T, Navita Bansal 25
Estimation of vitamin C, and -carotene Dr. Sweta Kumari and Veda
5. 28
content. Krishnan
6. Estimation of bioactive compounds by HPLC. Ms. VedaKrishnan 36
Estimation of proteins and amino acids. Dr. Suresh Kumar and Sweta
7. 43
Kumari
Protein characterization using different gel- Dr. Ranjeet R. Kumar, Suneha
8. 57
based proteomic tools Goswami and Shelly Praveen
Antioxidant enzymes and their activity assay Dr. Ranjeet R. Kumar, Suneha
9. 80
Goswami and Shelly Praveen
10. Agarose gel electrophoresis Dr. Vinutha T. 90
PCR-based cloning of a gene Dr. Aruna Tyagi and Kiswar
11. 100
Ali
Isolation of genomic DNA, Qualitative and Dr. Suresh Kumar and
12. 112
quantitative analyses of the DNA Archana Singh
Polymerase Chain Reaction, Isolation of plant Dr. Archana Singh
RNA, cDNA synthesis and RT-PCR expression
13. 122
profiling of genes using RT-PCR and
quantitative Real-Time PCR.
Southern blot analysis for determination of Dr. Archana Sachdev
14. 134
gene copy number
Measurement of pH and Preparation of reagents
Suresh Kumar
Division of Biochemistry, ICAR-Indian Agricultural Research Institute, New Delhi-

110012, India.
Introduction
The pH of a solution is a measure of the molar concentration of hydrogen ions [H+] in
the solution, and it is a measure of the acidity or basicity of the solution. The
symbol pH stands for "power of hydrogen" and numerical value for pH is just the
negative of the power of 10 of the molar concentration of hydrogen ions (H+). The ionic
product of water, Kw, is the basis for the pH scale. The total hydrogen ion concentration
from all sources is experimentally measurable and is expressed as the pH of the solution.
The pH scale designates the concentration of H+ (and thus of OH -) in any aqueous
solution in the range between 1.0 M H+ and 1.0 M OH-.
The term pH is defined by the expression:
Where the symbol p denotes “negative logarithm of”

Thus, pH can be defined as the –ve logarithm of the H+ concentration. For a
neutral solution at 25 C, in which the concentration of hydrogen ions is 1.0x10-7 M, the
pH can be calculated as follows:
Thus, the ionic product of water makes it possible to calculate the concentration
of H+, with given concentration of OH-, and vice versa. The value of 7 for pH of a neutral
solution is not an arbitrarily chosen figure; it is derived from the absolute value of the ion
product of water. A solution having pH greater than 7 is alkaline or basic, and the
concentration of OH- is greater than that of H+. A solution having a pH less than 7 is
acidic. Note that the pH scale is logarithmic, not arithmetic. For example: Two solutions
differ in pH by 1 unit, means that one solution has 10 times the H+ concentration of the
other; but it does not tell us the absolute magnitude of the difference. In other words, for
every 10‐fold change in concentration of the H+ ion (e.g. 0.1 to 1.0), the pH changes by 1
unit.
The pH of an aqueous solution can be approximately measured using various
indicator dyes, including litmus, phenolphthalein, and phenol red, which undergo color
changes as a proton dissociates from the dye molecule. Accurate determinations of pH in
the laboratory can be made with a glass electrode that is selectively sensitive to H+
concentration but insensitive to Na+, K+, and other cations. In a pH meter, the signal from
an electrode is amplified and compared with the signal generated by a solution of
accurately known pH. Measurement of pH is one of the most important and frequently
used procedures in biochemistry. The pH affects the structure and activity of biological
macromolecules; such as the catalytic activity of enzymes. Measurements of the pH of
blood and urine are commonly used in medical diagnoses.
1
Nearly everything around is an acid or a base, with the exception of water. Water
is neither acidic nor basic, rather it is neutral. The pH scale was developed to measure
how acidic or basic a substance is. This scale measures pH from 0 14. Acids have pH
values between 0 and 7, and bases have pH values between 7 and 14. Pure water is
neutral and has a pH of
exactly 7.
The pH of an aqueous
solution can be measured in a
variety of ways. The most
common way used now-a-
days is using a pH‐sensitive
glass electrode in a pH meter.
However, alternative methods
for measuring the pH of a
solution include use of (i)
litmus or Ph papers, (ii)
Indicator dyes, and (iii) a pH meter. Litmus is an extract from specific lichen which is
formed into a powder and then used in a chemical solution to treat paper, cut into strips,
and thus litmus paper is made. When litmus paper is dipped into a solution to check pH,
it changes colour depending on the pH of the solution. The colour of wetted litmus paper
is matched to a colour on a colour chart to infer a pH value. The pH paper is available
commercially for narrow pH ranges (e.g., 3.0 to 5.5 pH, 4.5 to 7.5 pH and 6.0 to 8.0 pH),
and fairly wide pH ranges of 1.0 to 11.0 pH. Also, three different types of litmus papers
(viz. blue, red and neutral) are available in the market. Blue litmus paper turns red in
acidic solutions, and remains blue in alkaline solutions. Red litmus paper turns blue in
alkaline solutions, and remains red in acidic solutions. However, neutral litmus
paper turns red in acidic solutions and turns blue in alkaline solutions. At neutral pH,
none of the litmus papers change colour. The litmus or pH paper is typically used for
preliminary and small volume measuring. It cannot be used for continuous monitoring of
a process. Though pH paper is fairly inexpensive, it may be affected by certain solutions,
which may interfere with the colour change.
Indicators are materials that change color when exposed to solution of different pH
values. They are used in acid-base titrations, and include Phenolphthalein, Methyl red,
and Bromothymol blue. These are also known as Universal indicators. Phenolphthalein
turns colorless in acidic solutions and pink in basic solutions. It adopts four different
states in aqueous solution: under strong acidic conditions, it exists in protonated form,
showing orange colour, under acidic conditions, its lactone form is colorless. Under basic
conditions, it shows singly deprotonated phenolate form and gives pink colour. However,
in strongly basic solutions (> 13.0 pH), it becomes completely colourless. Methyl red is a
dark red crystalline powder and it turns red in acidic solutions having pH < 4.4, yellow in
pH > 6.2, and orange in between. Bromothymol blue is mostly used in applications that
require measuring substances that would have a relatively neutral pH. A common use is
for measuring the presence of carbonic acid. It acts as a weak acid in solution; thus, it can
be in protonated (< pH 6.0, yellow) or deprotonated (> pH 7.6, blue) form.
A pH Meter is an instrument that measures the hydrogen-ion concentration or pH of

a solution, indicating its acidity or alkalinity. It measures the difference in electrical
potential between a pH electrode and a reference electrode, and display the result
2
converted into the corresponding pH value. Electrodes are the key parts: rod-like
structures usually made of glass, with a bulb containing the sensor at the bottom.
Frequent calibration with solutions of known pH, generally before each use, ensures the
accuracy of the instrument. The pH meters range from simple and inexpensive pen-like
devices to complex and expensive laboratory instruments with computer interfaces and
several inputs for indicator and temperature measurements to be entered to adjust for the
variation in pH caused by temperature. Specialty meters and probes are available for use
in special applications, harsh environments, etc.
For very precise work the pH meter should be calibrated before each measurement.
For normal use calibration should be performed at the beginning of each day. The reason
for this is that the glass electrode does not give a reproducible measurement over longer
periods of time. Calibration should be performed with at least two standard buffer
solutions that span the range of pH values to be measured. For general purposes buffers at
pH 4.00 and pH 10.00 are acceptable. The pH meter has one control (calibrate) to set the
meter reading equal to the value of the first standard buffer and a second control which is
used to adjust the meter reading to the value of the second buffer. A third control allows
the temperature to be set. Standard buffer sachets/solution can be obtained from
commercial suppliers. For more precise measurements, a three buffer solution calibration
is preferred. As pH 7 is essentially, a "zero point" calibration (zeroing or taring a scale or
balance), calibrating at pH 7 first, calibrating at the pH closest to the point of interest
(either 4 or 10) second, and checking the third point will provide a more linear accuracy.
Some meters allow a three-point calibration which is the preferred scheme for the most
accurate work. Higher quality meters will have a provision to account for temperature
coefficient correction, and high-end pH probes have temperature probes built in. After
each single measurement, the probe must be rinsed with distilled water to remove traces
of the solution adhering to the electrode,
blotted with a clean wipe to absorb any
remaining water.
Materials, Reagents and Solutions

• pH meter
• Wash bottle
• Distilled water
• pH solutions (pH 4.0, 7.0, 10.0)
• Kimwipes
Methods
Calibration of pH meter:
1. Before you begin to calibrate and use your pH meter you first need to turn the pH
meter on to warm up for an adequate time which generally takes around 30 minutes,
but check your pH meter’s operating manual for the exact time.
2. Take the electrode out of its storage solution and rinse it with distilled water under an
empty waste beaker. Once rinsed, blot dry with Kimwipes. Avoid rubbing the
electrode while wiping.
3. Since pH readings are temperature dependent, allow the standard buffers to reach to
the room temperature. Pour the buffers into individual beakers for calibration. Buffers
3
should be kept in a beaker for no longer than two hours. Do not pour used buffer back
into its original container.
4. Place the electrode in the buffer with a pH value of 7 and begin reading. Press the
calibrate button to begin reading the pH, allow the pH to stabilize by letting it sit for
approximately 1-2 minutes. Once the reading is stabilized, set the pH meter 7.0 by
pressing the measure button. If you stir your buffer before measuring be sure to stir
all other buffers and samples in the same way.
5. Rinse and dry the electrode with a lint-free Kimwipes.
6. Place the electrode in the buffer with a pH value 4.0 and begin reading by pressing
the measure button. Once the reading has stabilized, set the pH meter to pH 4.0.
7. Rinse and dry the electrode with a lint-free Kimwipes.
8. Use distilled water to rinse the electrode and dry with a lint-free Kimwipes.
9. If needed, calibrate the pH meter with standard pH buffer for 10.0.
10. Now, the pH meter is ready for taking measurement of pH of unknown samples.
Measurement of pH of a sample solution:
1. Rinse the electrode with distilled water under and blot dry with Kimwipes Place the
electrode in the sample, press the measure button and leave the electrode in the
sample for approximately 1-2 minutes.
2. Once the reading has stabilized, press the measure button and note down the pH nalue
of the sample.
3. Rinse the electrode with distilled water and blot dry with a lint-free Kimwipes. You
can take reading of other samples or store the pH meter for using next time.
4
Preparation of reagents, standard acids and alkali
Introduction
Commercially produced chemical reagents such as acids and ammonia are highly
concentrated solutions. For example, commercially available concentrated sulfuric acid
(H2SO4) is approximately 18 M. To prepare working solution of lower concentration for
qualitative analysis or titration, a calculated volume of the concentrated solution is taken
from the stock solution and then added to a specified volume of distilled water. However
the volume of the concentrated solution to be taken depends on the information provided
by the manufacturer on the label pasted on the bottle.
For example, the label on the bottle of concentrated H2SO4 the specific gravity is
mentioned to be 1.80. This means, the provided chemical is 1.80 times heavier than an
equal volume of H2O. One ml of the acid solution weighs 1.80 g or 1000 ml of the acid
solution weighs 1.80 x 1000 = 1800 g. Similarly, 98% by mass mentioned on the bottle
means: 98 g of the acid (solute) is present in 100 g of the solution. In other words, 100 g
of the acid solution contains 98 g of H2SO4. One liter (1000 ml) or 1800 g of the acid
solution contains (98/100) x 1800 = 1764 g pure H2SO4. Therefore, its mass concentration
= 1764 g/ L. Hence, its molar concentration, C = 1764 g/98 g = 18.0 Molar/L.
How to calculate molar concentration:
Generally, the original molar concentration (Co) of a chemical can be calculated using
the formula Co = (10 x P x d) / M, where P = Percent purity (% purity), d = density or
specific gravity of the chemical, and M = Molar mass.
Exercise 1: Calculate the molar concentration (Molarity) of commercial trioxonitric
acid ((HNO3) having specific gravity 1.42, purity 70.0%, and molar mass
is 63.0.
Answer: Co = ?, Given that P% = 70, d = 1.42, M = 63
Co = 10 x P x d = 10 x 70 x 1.42 =15.8 mole/L
M 63
Exercise 2: How will you prepare 40 mM hydrogen peroxide from 30% H2O2 solution?
Answer: The chemical dataset indicates 30% (w/w) = weight per weight, which means that the
solution contains 30 g H2O2 per 100 g of the solution.The density of the solution is 1.11 g/ml;
therefore, 100 g solution have a volume of 90.09 ml. So 1 liter will contain 333 g of H2O2, which
is a molar concentration of 9.79 M per L. Therefore, to prepare 40 mM solution take 4.09 ml of
30% H2O2 solution and make up the volume to 1 L with distilled water.
How to calculate volume of solution to be diluted:
For example, to prepare 500ml of 0.1 M H2SO4 from concentrated H2SO4 having Specific
gravity of 1.82, Purity = 97% and Molar mass = 98.
First of all calculate the concentration (Molarity) using the above formula.
Molarity = 10 x P x d = 10 x 1.82 x 97 = 18.01 M
M 98
Then, to calculate the amount of H2SO4 required to prepare 500 ml, 0.1 M H2SO4, the formula C1
x V1 = C2 x V2 can be used, where C1 = concentration of stock solution, V1 = volume of the of
5
stock solution required, C2 = concentration of the working solution to be made, and V2 = volume
of the of working solution to be made.
C1 = 18.01 M, V1 =?, C2 = 0.1 M, V2 = 500 ml
V1 = C2 x V2 = 0.1 × 500 = 2.78 ml
C1 18.01
Therefore, 2.78 ml of concentrated H2SO4 will be required to prepare 500 ml, 0.1 M
H2SO4.
6
Estimation of Carbohydrates
Vinutha T. and Veda Krishnan

110012, India.
Introduction
Carbohydrates are one of the most important components of the foods which deliver
energy. It may be present as isolated molecules or they may be physically associated or
chemically bound to other molecules. Individual molecules can be classified according to
the number of monomers that they contain as monosaccharides, oligosaccharides or
polysaccharides. Molecules in which the carbohydrates are covalently attached to
proteins are known as glycoproteins, whereas those in which the carbohydrates are
covalently attached to lipids are known as glycolipids. Some carbohydrates are digestible
by humans and therefore provide an important source of energy, whereas others are
indigestible and therefore do not provide energy. Indigestible carbohydrates form part of
a group of substances known as dietary fiber, which also includes resistant starch.
Consumption of significant quantities of dietary fiber has been shown to be beneficial to
human nutrition, helping reduceong the risk of certain types of cancer, coronary heart
disease, diabetes and constipation. As well as being an important source of energy and
dietary fiber, carbohydrates also contribute to the sweetness, appearance and textural
characteristics of many foods. It is important to determine the type and concentration of
carbohydrates in foods for a number of reasons. A large number of analytical techniques
have been developed to measure the total concentration and type of carbohydrates present
in foods. The carbohydrate content of a food can be determined by calculating the percent
remaining after all the other components have been measured: Carbohydrates (%) = 100
Moisture (%) Protein (%) Lipid (%) Mineral (%). Nevertheless, this method can
lead to erroneous results due to experimental errors in any of the other methods, therefore
so it is usually better to directly measure the carbohydrate content for accuracy in
estimation.
The critical parameters

To be considered during Sample preparation
The amount of preparation needed to prepare a sample for carbohydrate analysis depends
on the nature of the food being analyzed. Aqueous solutions, such as fruit juices, syrups
and honey, usually require very little preparation prior to analysis. On the other hand,
many foods contain carbohydrates that are physically associated or chemically bound to
other components, e.g., nuts, cereals, fruit, breads and vegetables. In these foods it is
usually necessary to isolate the carbohydrate from the rest of the food before it can be
analyzed. The precise method of carbohydrate isolation depends on the carbohydrate
type, the food matrix type and the purpose of analysis; however, there are some
procedures that are common to many isolation techniques. For example, foods are usually
dried under vacuum (to prevent thermal degradation), ground to a fine powder (to
enhance solvent extraction) and then defatted by solvent extraction. One of the most
commonly used methods of extracting low molecular weight carbohydrates from foods is
to boil a defatted sample with an 80% alcohol solution. Monosaccharides and
7
oligosaccharides are soluble in alcoholic solutions, whereas proteins, polysaccharides and
dietary fiber are insoluble. The soluble components can be separated from the insoluble
components by filtering the boiled solution and collecting the filtrate (the part which
passes through the filter) and the retentante (the part retained by the filter). These two
fractions can then be dried and weighed to determine their concentrations. In addition, to
monosaccharides and oligosaccharides various other small molecules may also be present
in the alcoholic extract that could interfere with the subsequent analysis e.g., amino acids,
organic acids, pigments, vitamins, minerals etc. It is usually necessary to remove these
components prior to carrying out a carbohydrate analysis. This is commonly achieved by
treating the solution with clarifying agents or by passing it through one or more ion-
exchange resins.
Clarifying agents
Water extracts of many foods contain substances that are colored or produce turbidity,
and thus interfere with spectroscopic analysis or endpoint determinations. For this reason
solutions are usually clarified prior to analysis. The most commonly used clarifying
agents are heavy metal salts (such as lead acetate) which form insoluble complexes with
interfering substances that can be removed by filtration or centrifugation. However, it is
important that the clarifying agent does not precipitate any of the carbohydrates from
solution as this would cause an underestimation of the carbohydrate content.
Ion exchange
Other than colorimetric analysis, many monosaccharides and oligosaccharides are polar
non-charged molecules and can therefore be separated from charged molecules by
passing samples through ion-exchange columns. By using a combination of a positively
and a negatively charged column it is possible to remove most charged contaminants.
Non-polar molecules can be removed by passing a solution through a column with a non-
polar stationary phase. Thus proteins, amino acids, organic acids, minerals and
hydrophobic compounds can be separated from the carbohydrates prior to analysis.
8
Estimation of Reducing Sugars by Nelson-Somogyi Method
Reducing sugars are frequently analyzed based on its free aldehyde and keto groups.
Some of the reducing sugars are glucose, galactose, lactose and maltose. In order for
oxidation to occur, the cyclic form must first open the ring form to give reactive
aldehyde. So any sugars that contain a hemiacetal will be a reducing sugar, but glycosides
which are acetals are not reducing sugars. Ketoses can also be reducing sugars because
they tautomerise to aldose via an enediol. The Nelson-Somogyi method is one of the
classical and widely used methods for the quantitative determination of reducing sugars.
Principle
This procedure is a modification of Somogyi's titrometric method for use with
colorimeter and employs copper and arsenomolybdate reagent. The reducing sugars such
as glucose, galactose, lactose and maltose when heated with alkaline copper tartrate,
reduce the copper from cupric to cuprous state and the cuprous oxide is formed. This
cuprous oxide when treated with arsenomolybdate, the reduction of molybdic acid to
molybdenum blue takes place. The blue colour developed can be compared with the
standards of known quantity in a colorimeter at 520nm. The range of test is 5 to 600µg of
sugar.
Reagents
1. Arsenomolybadate reagent (Nelson’s reagent): 25g ammonium molybdate is
dissolved in 450 ml of water and 21 ml of concentrated sulphuric acid is slowly added
with stirring. Then 3 g of sodium hydrogen arsenate dissolved in 25 ml distilled water,
is poured slowly with constant stirring. The solution is incubated at 370C for 24 hrs.
filtered and stored in amber coloured reagent bottle.
2. Copper reagent
(a) Copper reagent A: 25 g of anhydrous sodium carbonate, 25 g of sodium potassium
tartarate (Rochelle salt) and 20 g sodium bicarbonate are dissolved in about 700 ml
of water, finally add 200 g of anhydrous sodium sulphate and dilute the solution to
one litre, and allow to keep it for two days at room temperature. Collect the clean
supernatant by filtering.
(b) Copper reagent B: 15 g CuSO4.5H2O is dissolved in 100 ml water. Add 1 drop of
H2SO4.
(c) Working copper reagent C: Prepare fresh before use by mixing 25 ml of copper
reagent A and 1 ml of copper reagent B.
3. Glucose standard (Stock solution, 1 mg per ml) : Dissolve 100 mg of glucose in
100 ml of distilled water.Working standard solution (100µg glucose per ml): From
the standard stock solution of glucose, pipette 10 ml in 100 ml volumetric flask and
make up the volume with distilled water. The working standard of glucose will be
100 µg per ml.
Procedure
1. Weigh 100 to 500 mg of sample and extract the material with hot 80 % ethanol in
mortar and pestle.
2. Collect the supernatant and evaporate the ethanol on a water bath at 800C. Add 10 ml
water and dissolve the material.
9
3. Pipette out aliquots of 0.1 to 2 ml in separate test tubes.
4. Also pipette out 0.2, 0.4, 0.6, 0.8 and 1 ml of working standard glucose in another set
of test tubes. Make the volume to 2 ml with distilled water. Pipette out 2 ml of distilled
water in a separate tube which can be used as blank.
5. Add 2 ml of alkaline working copper reagent C to all the tubes. Mix well. Heat the
content in a boiling water bath for 10 min and cool to room temperature.
6. Add 1 ml of arsenomolybdate reagent to all the tubes. Mix well and make the volume
to 10 ml with water. Read the blue colour at 520 nm in colorimeter/spectrophotometer.
Although the colour is stable determine the absorbance at a fixed time after the
addition of arsenomolybdate reagent. Calculate the amount of reducing sugar from
standard curve.
Calculation
Absorbance corresponds to 0.1 ml of test sample = Y mg of glucose

10 ml contains = (Y/0.1) × 10 mg of glucose
= % of reducing sugars
NOTE: Alcohol insoluble residue contains macromolecules such as starch, pectin, gums,
hemicellulose, cellulose, lignin, proteins, insoluble ash, and minerals.
References
Somogyi M (1945) A new reagent for determination of sugars. J. Biol. Chem. 160: 61.
Nelson N (1944) A photometric adoption of the Somogyi's method for determination of
glucose. J. Biol. Chem. 153: 375.
10
Estimation of Reducing Sugars by Dinitrosalicylic Acid (DNS) method
Sugars that contain aldehyde groups that are oxidized to carboxylic acids are classified as
reducing sugars. All monosaccharides are reducing sugars, along with some
disaccharides, oligosaccharides, and polysaccharides. The monosaccharides can be
divided into two groups: the aldoses, which have an aldehyde group, and the ketoses,
which have a ketone group. Ketoses must first tautomerize to aldoses before they can act
as reducing sugars. The common dietary monosaccharides galactose, glucose and
fructose are all reducing sugars. Accurate determination of reducing sugars is necessary
for various applications in food, agriculture and health industry. The 3,5-dinitrosalicylic
acid (DNS) method is one of the classical method for estimation.
Principle
The 3,5-dinitrosalicylic acid (DNS) method for determining reducing sugar concentration
was developed by Sumner and later modified by Miller. When alkaline solution of 3,5-
dinitrosalicylic acid reacts aldehyde groups on the with reducing sugars (eg. Glucose,
lactose.) it is converted into 3-amino-5-nitrosalicylicacid which gives orange color which
can be read at wavelength of light 540nm using spectrophotometer. Water is used up as a
reactant and oxygen gas is released during the reaction. Intensity of the colour is an index
of reducing sugar.
(Chemical formula/reaction)
Equipment
Spectrophotometer capable of measuring absorbance in the 540 nm region.

Cuvettes for spectrophotometer
Water bath 100°C
Reagents
Glucose standard solutions at the concentration ranging from 0.6 µmol/ml to 4.00
µmol/ml
0.05 M acetate buffer pH 4.8
DNS reagent
10 g 3,4-dinitrosalicyclic acid
403 g potassium sodium tartrate tetrahydrate
16 g NaOH (anhydrous)
11
Preparation
In 1000 ml beaker dissolve 10 g of 3,4-dinitrosalicyclic acid in 200 ml H2O. Followed

by continuous stirring slowly add a solution of NaOH dissolved in 150 ml distilled
water.
• Incubate mixture in 50°C with stirring to obtain a clear solution.
• In small portions add 403 g of potassium sodium tartrate tetrahydrate.
• Filter mixture using paper filter and make up the volume to 1000 ml with water.
• Store in dark glass bottle at temperature below 20 °C.
Procedure
 Prepare glucose standard solutions in 0.05 M acetate buffer (pH 4.8) ranging from
0.6-4.00 µmol/ml.
 Add 1 ml of each standard to separate tubes. To the tubes used as the blanks, add 1
ml of 0.05 M acetate buffer (pH 4.8).
 Prepare the unknown samples in an appropriate dilution.
 To each tubes, add 1 ml of 0.05 M acetate buffer (pH 4.8) and mix.
 Add 3 ml DNS reagent to all the test tubes and mix well.
 Place the tubes in boiling water for 5 minutes.
 Cool the tubes to room temperature and measure the absorbance at 540 nm.
Calculation
The amount of carbohydrate in 1 g sample = ----- mg/dl dilution factor 100
Dilution factor = final volume/initial (aliquot) volume
References
Sumner JB (1925) A more specific reagent for the determination of sugar in urine. J.
Biol.Chem. 65: 393 395.
Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar.
Anal. Chem. 31: 426 428.
12
Determination of Total Carbohydrate by Anthrone Method
Carbohydrates are the important components of storage and structural materials in the
plants. They exist as free sugars and polysaccharides. The basic units of carbohydrates
are the monosaccharides which cannot be split by hydrolysis into more simpler sugars.
The carbohydrate content can be measured by hydrolyzing the polysaccharides into
simple sugars by acid hydrolysis and estimating the resultant monosaccharides. Anthrone
reagent is most commonly employed for this purpose.
Principle
Carbohydrates are first hydrolysed into simple sugars using dilute hydrochloric acid. In
hot acidic medium glucose is dehydrated to hydroxymethyl furfural. This compound
forms with anthrone a green coloured product with an absorption maximum at 630 nm.
Reagents
2.5 N HCl
Anthrone reagent: Dissolve 200 mg anthrone in 100 ml of ice-cold 95% H2SO4.
Prepare fresh before use.
Standard glucose: Stock—Dissolve 100 mg in 100 ml water.
Working standard: 10 ml of stock diluted to 100 ml with distilled water. Store at 4 C
after adding a few drops of toluene.
Procedure
 Weigh 100 mg of the sample into a boiling tube.

 Hydrolyse by keeping it in a boiling water bath for three hours with 5 ml of 2.5 N HCl
and cool to room temperature.
 Neutralize it with solid sodium carbonate until the effervescence ceases.
 Make up the volume to 100 ml and centrifuge.
 Collect the supernatant and take 0.5 and 1 ml aliquots for analysis.
 Prepare the standards by taking 0, 0.2, 0.4, 0.6, 0.8 and 1 ml of the working standard.
 ‘0’ serves as blank.
 Make up the volume to 1 ml in all the tubes including the sample tubes by adding
distilled water.
 Then add 4 ml of anthrone reagent.
 Heat for eight minutes in a boiling water bath.
 Cool rapidly and read the green to dark green colour at 630 nm.
 Draw a standard graph by plotting concentration of the standard on the X-axis versus
absorbance on the Y-axis.
 From the graph calculate the amount of carbohydrate present in the sample tube.
Calculation
Amount of carbohydrate present in 100 mg of the sample= mg of glucose/Volume of test

sample×100
NOTE: Cool the contents of all the tubes on ice before adding ice-cold anthrone reagent.
13
References
Clegg KM (1956) The application of anthrone reagent to the estimation of starch in
cereals. J. Sci. Fd. Agric. 7: 40 44.
Hodge JE, Hofreiter BT (1962) In: Methods in Carbohydrate Chemistry (Eds. Whistler,
R.L. and BeMiller. J.N.) Acad. Press, New York.
14
Determination of Amylose
Starch quality is majorly governed by its components – amylose and amylopectin. These
are the quality matrics which indirectly affect the eating quality of rice. Amylose is a
linear chain of (1)-linked α-D-glucopyranosyl units and many amylose molecules have
very few α-D-(1) branches. Amylose has a right-hand helix linear structure without
bends. The inside of the helix is lipophilic where there are only hydrogen atoms. On the
outside, there are hydrophilic hydroxyl groups. Alpha-D-(1) branches may occur once in
every 180-320 units, or 0.3- 0.5% of the linkages. Amylose in starch is responsible for the
formation of a deep blue color in the presence of iodine. The iodine molecule slips inside
of the amylose coil. Iodine is not very soluble in water; therefore the iodine reagent is
made by dissolving iodine in water in the presence of potassium iodide. This makes a
linear tri-iodide ion complex with is soluble. The tri-iodide ion slips into the coil of the
starch causing an intense blue-black color – amylose-iodine complex. This method uses
common inexpensive chemicals and does not require any expensive instrumentation. The
details of this simplified method are presented below.
Principle
The iodine is adsorbed within the helical coils of amylose to produce a blue-coloured
complex which is measured colorimetrically at 590 nm.
Reagents
Distilled ethanol
1 N NaOH.
0.1% phenolphthalein.
Iodine reagent: Dissolve 1 g iodine and 10 g KI in water and make up to 500 ml.
Standard: Dissolve 100 mg amylose in 10 ml 1 N NaOH; make up to 100 ml with
water.
Procedure
 Weigh 100 mg of the powdered sample, and add 1 ml of distilled ethanol. Then add
10 mL of 1 N NaOH and leave it overnight.
 Make up the volume to 100 ml.
 Take 2.5 ml of the extract, add about 20 ml distilled water and then three drops of
phenolphthalein.
 Add 0.1 N HCl drop by drop until the pink colour just disappears.
 Add 1 ml of iodine reagent and make up the volume to 50 ml and read the colour at
590 nm.
 Take 0.2, 0.4, 0.6, 0.8 and 1 ml of the standard amylose solution and develop the
colours in the case of sample.
 Calculate the amount of amylose present in the sample using the standard graph.
 Dilute 1 ml of iodine reagent to 50 ml with distilled water for a blank.
Calculation
Absorbance corresponds to 2.5 ml of the test solution = Y mg amylose
100 ml contains = (2.5/Y) × 100 mg amylose = % amylose.
15
Notes: (i) The sample suspension may be heated for 10 min in a boiling water-bath
instead of overnight dissolution.
(ii) The amount of amylopectin is obtained by subtracting the amylose content
from that of starch.
References
McCready RM, Guggolz J, Siliviera V, Owens HS (1950) Determination of starch and
amylose in vegetables. Anal. Chem., 22: 1156 1158.
16
Estimation of Resistant Starch
Starch is the major energy reserve in plants and can be found in grains, roots, tubers,
stems, leaves, and fruits. Starch consists of two major glucans: amylose and amylopectin.
Amylose is an essentially linear polysaccharide with -1 4 linked D-glucopyranose
units and a few branches of -1 6 linkages, whereas amylopectin is a highly-branched
polysaccharide consisting of short linear-chains connected by about 5% -1 6 branch
linkages. Starch is a major energy source for humans and animals. After being ingested,
starch is hydrolyzed to glucose by amylolytic enzymes in the digestive tract. Glucose is
then absorbed in the small intestine to be used for energy. A portion of starch, known as
resistant starch (RS), cannot be hydrolyzed by enzymes in the small intestine and is
passed into the large intestine for fermentation by gut microflora. RS is categorized as a
type of dietary fiber because it can provide similar physiological benefits to humans and
animals as other dietary fibers. Health benefits of RS include: lowering postprandial
plasma glucose concentration and insulin response; increasing insulin sensitivity and
preventing Type-2 diabetes; improving serum lipid profile and preventing obesity; and
improving colon health. RS can be classified into five different types: physically
inaccessible starch (RS1); raw granular starch with the B- or C-type polymorph (RS2);
retrograded starch (RS3); chemically modified starch (RS4); and amylose-lipid complex
(RS5). When amylose forms single-helical complex with lipids, it becomes resistant to
enzymatic hydrolysis
Principle
The RS content in the rice genotypes will be determined based on an enzymatic method
(AOAC, 2002). The samples will be incubated with pancreatic α-amylase and
amyloglucosidase (AMG) for 16 h at 37°C. During this time the non-resistant starch will
be solubilized and hydrolyzed to glucose by the combined action of these two enzymes.
The reaction would be terminated by the addition of an equal volume of ethanol and the
RS will be recovered as a pellet on centrifugation. This will later be washed twice with
ethanol (50% v/v), and again centrifuged. The RS in the pellet will be dissolved in 2 M
KOH by vigorously stirring on an ice-water bath. This solution would be neutralized with
acetate buffer and the starch will be quantitatively hydrolyzed to glucose with AMG. The
glucose would be quantified with glucose oxidase/peroxidase reagent (GOPOD), which
would be giving a measure of the RS content of the sample.
Materials
Powdered rice grain,

Shaking water bath,
Test tubes,
Magnetic stirrer,
Centrifuge.
Reagents
Sodium maleate buffer (0.1 M, pH 6),

Sodium acetate buffer (1.2 M, pH 3.8 and 0.1 M, pH 4.5),
Potassium hydroxide (2M),
Aqueous ethanol (Industrial Methylated Spirits or IMS).
17
Reagents
Sodium maleate buffer (0.1 M, pH 6) – Dissolve 23.2 g of maleic acid (Sigma cat. no.
M0375) in 1600 mL of distilled water and adjust the pH to 6.0 with 4 M (160 g/lit)
sodium hydroxide. Add 1.47 g of calcium chloride dehydrate (CaCl2.2H2O) and
dissolve. Adjust the volume to 2 L. Stable for 12 months at 4°C.
Sodium acetate buffer (1.2 M) – Add 69.6 ml of glacial acetic acid (1.05 g/ml) to 800
ml of distilled water and adjust to pH 3.8 using 4 M sodium hydroxide. Adjust the
volume to 1 lit with distilled water. Stable for 12 months at room temperature.
Sodium acetate buffer (0.1 M) – Add 5.8 ml of glacial acetic acid to 900 mL of
distilled water and adjust to pH 4.5 using 4 M sodium hydroxide. Adjust the volume to
1 L with distilled water. Stable for 2 months at 4°C.
Potassium hydroxide (2M) – Add 112.2 g KOH to 900 ml of deionized water and
dissolve by stirring. Adjust volume to 1 L. Store in a sealed container. Stable for >2
years at room temperature.
Aqueous ethanol (or IMS) (approx. 50% v/v) – Add 500 ml of ethanol (95% v/v or
99% v/v) or industrial methylated spirits (IMS; denatured ethanol; ~ 95% v/v ethanol
plus 5% v/v methanol) to 500 ml of H2O. Store in a well-sealed bottle. Stable for > 2
years at room temperature.
Procedure
1. Weigh about 100 mg of the sample individually into screw cap tube, add 4.0 ml of
pancreatic α-amylase solution (10 mg/ml) containing AMG (3 U/ml) and subsequently
incubate at 37°C with continuous shaking (200 strokes/min) for exactly 16 h.
CRITICAL STEP: Horizontal shaking is better and it’s critical that samples should be
immersed in solution.
2. Disperse 4.0 ml of 99% (IMS)/alcohol into tube with vigorous stirring and then
centrifuge at 1,500g for 10 min.
3. The supernatants would be decant and pellets would be re-suspended in 2 mL of 50%
IMS/alcohol, vortex vigorously and add 6 ml of 50% IMS/alcohol, vortex and again
centrifuge at 1,500g for 10 min.
4. Above step 4 is repeated twice.
CRITICAL STEP: Complete removal of alcohol from the tubes is necessary
5. The pellet would be kept for resistant starch determination whereas the centrifuged
washings will be combined with the original decanted supernatant and used for
solubilized starch determination. The pellet would be re-suspended with 2 ml of 2 M
KOH.
6. After continuous stirring for 20 min in an ice bath, 8 mL of 1.2 M sodium acetate
buffer (pH 3.8) and 0.1 ml of AMG (3300 U/ml) would be added and the tubes would
be placed in a water bath at 50°C for 30 min.
CRITICAL STEP: Proper stirring by placing in ice is critical, pellet should be
completely dissolved.
7. At the end of the treatment, the solution would be brought to a total volume of 100 ml
(optional) with distilled water, mixed and centrifuged at 1500 rpm for 10 min.
18
8. 0.1 ml aliquot of supernatant would be treated with 3.0 ml of D-glucose (oxidase-
peroxidase) assay kit (GOPOD) reagent (Megazyme, Ireland), vortex-mixed and kept
at 50°C for 20 min.
CRITICAL STEP: Working GOPOD can be stored in 4°C, if turned pinkish should be
discarded.
9. Blank (0.1 ml of 100 mM sodium acetate buffer pH 4.5) and 3.0 mL of GOPOD and
glucose standards 0.1 mL of D-glucose (1 mg/ml) and 3.0 ml of GOPOD, would be
incubated concurrently.
10. The absorbance would be read with a spectrophotometer at 510 nm. The total starch
contents are calculated as the sum of digestible starch and resistant starch.
Calculation
Resistant Starch (g/100 g sample) (samples containing < 10% RS):
= ΔE F 10.3/0.1 1/1000 100/W 162/180
= ΔE F/W 9.27
Where:
ΔE = absorbance (reaction) read against the reagent blank.
F = conversion from absorbance to micrograms (the absorbance obtained for 100 µg of
D-glucose in the GOPOD reaction is determined and F = 100 (µg of D-glucose)
divided by the GOPOD absorbance for this 100 µg of D-glucose.
100/0.1 = volume correction (0.1 ml taken from 100 ml).
1/1000 = conversion from micrograms to milligrams.
W = dry weight of sample analyzed = “as is” weight [(100 moisture content)/100]
100/W = factor to present RS as a percentage of sample weight.
162/180 = factor to convert from free D-glucose, as determined, to a hydro-D-glucose as
occurs in starch.
10.3/0.1 = volume correction (0.1 ml taken from 10.3 ml) for samples containing 0-10%
RS where the incubation solution is not diluted and the final volume is ~ 10.3
ml.
References
AACC International (2000) Approved methods of the American Association of Cereal

Chemists. St. Paul, MN: AACC, Inc. Yang CZ, Shu XL, Zhang LL, Wang XY, Zhao HJ,
Ma CX, Wu DX. (2006) J. Agric. Food. Chem. 2: 523 528.
Zhou H, Wang L, Liu G, Meng X, Jing Y, Shu X, Kong X, Sun J, Yu H, Smith SM, Wu
D and Li J (2016) Critical roles of soluble starch synthase SSIIIa and granule-bound
starch synthase Waxy in synthesizing resistant starch in rice. Proc. Natl. Acad. Sci. 113:
12844–12849.
Perera A, Meda V, and Tyler RT (2010) Resistant starch: A review of analytical
protocols for determining resistant starch and factors affecting the resistant starch content
of foods. Food Res. Int. 43: 1959–1974.
19
Extraction of Oil and Estimation of its
Characteristic Features
Vinutha T. and Navita Bansal

110012, India.
Introduction
Fats and oils belong to a group of biological substances called lipids. They are
constructed of building blocks called triglycerides resulting from combination of one unit
of glycerol and three units of fatty acids. They are insoluble in water but soluble in most
organic solvents. They have lower densities than water, and may have consistencies at
ambient temperature of solid, semi-solid or clear liquid. When they are solid-appearing at
normal temperature, they are referred to as “fats” and when they are liquid at the ambient
temperature, they are called oils.
Method of extraction and estimation of oil
Extraction of oil from an oil-seed is generally carried out using the following two
methods:
(a) Soxhlet method
(b) Cold percolation method
(a) Soxhlet method
Principle
The principle is based on the extraction of oil using non-polor solvent viz., ether, hexane,
petroleum ether (40-60 oc), chloroform:methanol (2:1). And it involves repeated
extraction of oil. The solvent is then distilled off completely. The oil is dried, weighed
and the % of oil is calculated.
Apparatus
1. soxhlet extractor assembly
2. Absorbent cotton
3. Whatman filter paper/ no?
Reagent
Hexane or diethyl ether/petroleum ether (40-60 oc).

Procedure
1. Fold a piece of filter paper and make into a sample packet in such a way to hold the
seed powder (2-5g) depending on oil content.
2. Place the sample packet into the extractor of soxhlet apparatus, after placing some
cotton at the bottom. A piece of cotton is placed at the top to evenly distribute the
solvent as it drops on the sample during extraction.
20
3. Add organic solvent, two and a half times the capacity of the extractor and extract
oil for a period of six hours, or for a longer period till the solvent in the extractor
becomes colourless.
4. Put off the heaters and allow to cool . Flash evaporate the solvent or evaporate the
ether on a water bath until no odour of ether remains. Keep it in oven at 70 degree
for 10 min. Cool at room temperature.
5. Weigh the flask after removing moisture. Repeat heating until constant weight is
recorded.
Calculations
% Oil in the sample = Weight of oil (g) x 100/Weight of sample (g)
Suggested Reading
Association of official Analytical Chemists, 1965. Methods of Analysis.
(b) Percolation method
Principle
The extraction of oil from the sample involves the use of non-polar solvent viz.hexane,
petroleum ether (40-60o C) which percolate through a column containing anhydrous
sodium sulphate. The oil gets eluted by the solvent and is collected in a conical flask.
Reagents
1. Anhydrous sodium sulphate

2. CCl4 or hexane or petroleum ether (40-60oC) or solvent ether.
Procedure
1. Grind 1-2g of seed material to a fine powder with the help of 20-30g of anhydrous
sodium sulphate, mix well.
2. Plug the percolator by putting cotton glass wool at the bottom of it . Put
approximately 2g of sodium sulphate (anhydrous) over the plug.
3. Pack the powdered material slowly inside the percolator carefully. Ensure that
whole of the material is packed properly, pack little cotton over the material and
cover with a layer of anhydrous sodium sulphate.
4. Add 10ml solvent in the mortas and pestle to ensure complete removal of the
material and transfer it into the percolator.
5. Keep a flask below the percolator and fill the percolator with the solvent.
6. Let the solvent tickle down under gravity. The solvent will flow down after
extracting oil from the material.
7. Add more solvent, to fill the percolator and allow it to pass through the column.
Repeat this thrice.
21
8. Flash evaporate or distill of the solvent using water bath. When 5ml of solvent is
left in the flask (Round bottom in case of flash evaporator/distillation), remove it
and transfer into a weighed conical flask and keep in the oven till the solvent is
evaporated. Weigh to constant weight after keeping in desiccator for few minutes.
Calculations:
Weight of flask + Glass bead =Xg
Weight of flask + Glass bead + oil = Y g
Weight of oil = Y-X g
% oil = (Y-X) 100
Wt. of the material
References
Kartha, A.R.S and Sethi, A.S (1957). Ind.J.Agric.Sci. 27:211.
22
Estimation of Acid Value of Oil
Introduction:
The acid value of oil is dependent on the amount of free fatty acids present. This in turn
is dependent on the degree of hydrolysis of oil or nature of processing which the oil may
have undergone. Refined oils are often referred to as neutralized i.e they have low acid
value. Raw and crude oils are naturally hydrolysed and consequently have a high acid
value.
Definition
The acid value is the number of milligrams of potassium hydroxide required to neutralize
the free fatty acids present in 1 g of fat or oil.
Principle
Free fatty acids are usually present in small quantity in oil along with the triglycerides,
the proportion increase during storage. The fat/oil on storage may become rancid due to
peroxide formation by microorganisms in presence of atmospheric oxygen. The amount
of free fatty acids gives an indication of age and quality of fat. The free fatty acids in oil
are estimated by titrating it against standard potassium hydroxide in the presence of
phenolphthalein indicator.
Reagents
1. 0.1N KOH
2. Neutral solvent: Mix equal volume of 95% ethanol and solvent ether. Acid or
alkali is added to make it neutral depending on the pH.
3. 0.1N potassium hydrogen phthalate for standardization of KOH or 0.1N oxalic
acid.
4. 1% phenolphthalein as indicator.
Procedure
1. Accurately weigh 1-10 g oil in a clean dry 100 ml conical flask.

2. Add 25 ml of neutral solvent solution and add few drops of phenolphthalein and
warm it.
3. Titrate the warm content against 0.1N potassium hydroxide.
4. Shake constantly until a pink colour which persists for 30 sec is obtained.
5. Run a blank following the similar procedure as for the test sample.
23
Calculations
Acid value (AV) = (Titre value- blank value) Normality of KOH 56.1
Weight of the sample
The free fatty acid (FFA) is calculated as oleic acid equivalent using the
equation
% FFA = AV/1.99 or 1 ml of N/10 KOH = 0.0282 g of oleic acid (on the basis
of oleic acid)
Say Normality of KOH is in
i.e N gm equivalents/100 ml
Since acidity of KOH is 1
= N moles/1000 ml
=N 56.1 g/1000 ml
=N 56.1 1000mg/1000 ml
Or 1000ml contains = N 56.1 1000 mg of KOH
Say net titre value is V, and then amount of KOH consumed in the reaction is:
N 56.1 1000 V mg
1000
Say this value has been obtained for W g of sample
Acid value for 1 g of sample will be = (V N 56.1) / W
References
Association of official analytical chemists (1975). Official methods of analysis, 12th Ed.
P. 492, Washington D.C.
24
Estimation of Iodine Value of an Oil Sample
Vinutha T, Navita Bansal
Introduction
In chemistry, it is the mass of iodine in grams that is consumed by 100 gm of a chemical
substance. An iodine solution is violet in color and any chemical group in the substance.
That reacts with iodine will make the colour disappear at a precise concentration. The
amount of iodine solution thus required to keep the solution viole is a measure of the
iodine sensitive reactive groups.
Iodine value is a measure of unsaturation. The higher the iodine value, the
higher the level of unsaturation and more reactive the oil is. Non – drying oils have
low iodine values (<125) whereas drying oils have high iodine values (>150). Oils
with iodine value between 125 and 150 are known as semi-drying oils. Solid fats have
low iodine value (typically<60).
If oil consists of triglycerides with a high percentage of unsaturated fatty acids

such as linolenic acid then that oil will be reactive and is known as drying oil. This means
that when applied to a non – porous surface it will dry to a film in a certain time. For
example linseed oil contains 50-60% linolenic acid (18:3) and will usually dry in less
than 4 days without additional processing or the incorporation of driers. Towards the
other end of the scale is rapeseed oil. This contains 52-62% oleic acid (18:1). Therefore
oil is not as reactive, so will not dry to a film when exposed to air . It is consequently
classified as non-drying oil.
Main fatty acid present Iodine value Oil drying
Linseed Linoleic (51-60%) 175-195 Oil drying
Safflower Linoleic (70-80%) 135-148 Semi-drying
Soyabean Linoleic (50-56%) 129-143 Semi-drying
Sunflower Linoleic (60-70%) 112-138 Semi- drying
Rapeseed L/E Oleic (56-62%) 100-125 Non-drying
Rapeseed H/E Erucic (45-55%) 100-120 Non-drying
Arachis Oleic (35-45%) 85-105 Non-drying
Castor Ricinoleic 82-90 Non-drying
Drying and semi-drying oils find uses in surface coating applications where drying
properties are essential e.g. paints, resins, printing inks.
Non-Drying oils, generally find uses in applications where oxidation is undesirable e.g.
lubricants, leather dressings.
Definition
Iodine value of oil is defined as number of grams of iodine absorbed by 100 gm of oil or
fat.
25
Principle
This contain oil both saturated and unsaturated fatty acids such as oleic and linoleic acids
as glyceryl esters (triacyl glycerols). Iodine gets incorporated into the fatty acid chain
wherever the double bond exists to form addition (saturated) compounds.
CH3 (CH2)7 CH = CH (CH2)7COOH + IBr
CH3 (CH2)7 CH-CH (CH2)7 COOH
Br I
IBr + KI = KBr+ I2
2Na2S2O3 + I2 = 2NaI + Na2S4O6 (Colourless)
Present experiment makes use of iodine monobromide (Hanus solution) to saturate a
known weight of oil. The unreacted iodine monobromide is then reacted with potassium
iodide which converts it into I2. The I2 concentration is then determined by titration with
standard sodium thiosulfate using starch as an indicator.
Reagent
1. 0.1 N sodium thiosulfate

2. 15% potassium iodide
3. 0.3N Hanus solution(IBr) in glacial acetic acid
4. 1% starch solution
5. 0.1 N potassium dichromate ( required for standardization of sodium thiosulfate)
Procedure
1. Weigh 50-100 mg oil into an iodine flask and dissolve in 25 ml of carbon

tetrachloride.
2. Add 25 ml of Hanus solution from a burette and keep the flask in dark for 45 min.
with occasional shaking.
3. Add 10 ml of 15% potassium iodide solution mix thoroughly and add 25 ml of
water, to washing much down any free iodine on the stopper.
4. Titrate against 0.1 N sodium thiosulfate till the colour of the titre becomes pale
yellow. From this point onwards colour fades very slowly from pale yellow to
colourless.
5. To get a definite colour change at the end point, 1 ml of starch indicator is added
in the solution which turns it blue black. Titration is then continued till the end
point is obtained when colour suddenly changes from blue black to colourless.
6. A blank determination is carried out without oil, using exactly the same quantity
of carbon tetrachloride and keeps it for the same duration in Hanus solution and
determine amount of used IBr.
Calculation
Iodine value = (V2 – V1) Normality of thiosulphate 12.69
Weight of oil
V2 = Volume of thiosulphate used for blank
V1 = Volume of thiosulphate used for oil sample.
26
Note:
1. One ml of 1.0 N Na2S2O3 = 0.1269 g of iodine.
2. Starch forms an insoluble complex with iodine. The titer problem precludes the
addition of the indicator at the start of the titration and this is why the indicator is
to be added just prior to the end point when the colour of the solution has
changed to pale yellow.
References
Association of official analytical chemists (1975). Official methods of analysis, 12 th Ed.
P. 488, Washington D.C.
27
Estimation of vitamin A and C content
Sweta Kumari and Veda Krishnan

110012, India.
Estimation of vitamin A
Introduction
Carotenoids, also called tetraterpenoids, are organic pigments that are produced by plants
and algae, as well as several bacteria and fungi. The only animals known to produce
carotenoids are aphids and spider mites, which acquired the ability and genes from fungi.
There are over 600 known carotenoids; they are split into two
classes, xanthophylls (which contain oxygen) and carotenes (which are purely
hydrocarbons, and contain no oxygen). All are derivatives of tetraterpenes, meaning that
they are produced from 8 isoprene molecules and contain 40 carbon atoms. In general,
carotenoids absorb wavelengths ranging from 400 550 nanometers (violet to green light).
This causes the compounds to be deeply colored yellow, orange, or red.
One of the most important physiological functions of carotenoids in human
nutrition is to act as pro-vitamin A (vitamin A precursors). Pro-vitamin A carotenoids
support the maintenance of healthy epithelial cell differentiation, normal reproductive
performance, and visual functions. Additionally, non-provitamin A carotenoids (e.g.
lutein, astaxanthin, zeaxanthin and lycopene) also play an important role in human health
as biological antioxidants, protecting cells and tissues from the oxidative damaging
effects of free radicals and singlet oxygen
β-carotene is strongly colored red-orange pigment abundant in plants and fruits. It
is a member of the carotenes, which are terpenoids (isoprenoids), synthesized
biochemically from eight isoprene units and thus having 40 carbons. Among the
carotenes, β-carotene is distinguished by having beta-rings at both ends of the molecule.
Isolation of β-carotene from fruits abundant in carotenoids is commonly done
using column chromatography. The separation of β-carotene from the mixture of other
carotenoids is based on the polarity of a compound. β-carotene is a non-polar compound,
so it is separated with a non-polar solvent such as hexane, petroleum ether. Being
highly conjugated, it is deeply colored, and as a hydrocarbon lacking functional groups, it
is very lipophilic.
Structure of β-carotene
28
Provitamin A activity
Plant carotenoids are the primary dietary source of provitamin A worldwide, with β-
carotene as the best-known provitamin A carotenoid. Others include α-carotene and β-
cryptoxanthin. Carotenoid absorption is restricted to the duodenum of the small intestine
and dependent on class B scavenger receptor (SR-B1) membrane protein, which are also
responsible for the absorption of vitamin E (α-tocopherol). One molecule of β-carotene
can be cleaved by the intestinal enzyme β,β-carotene 15,15'-monooxygenase into two
molecules of vitamin A. Absorption efficiency is estimated to be between 9 and 22%.
The absorption and conversion of carotenoids may depend on the form of β-carotene
(e.g., cooked vs. raw vegetables, or in a supplement), the intake of fats and oils at the
same time, and the current stores of vitamin A and β-carotene in the body.
Dietary sources
Beta-carotene is found in many foods and is sold as a dietary supplement. β-carotene
present in yellow to orange color fruits and vegetables. Rich sources are yellow and
orange fruits, such as cantaloupe, mangoes, pumpkin, papayas, and orange root
vegetables such as carrots and sweet potatoes. The color of β-carotene is masked
by chlorophyll in green leafy vegetables such as spinach and kale. Carrots are one of the
best sources of beta-carotene and its content has been reported up to 28 mg/100g. One
cup of carrot juice has more beta-carotene than a cup of any other food. One baked sweet
potato and 1 cup of canned pumpkin and cooked spinach are the only foods that supply
more beta-carotene than 1 cup of cooked carrots.
Conversion factors
Since 2001, the US Institute of Medicine uses retinol activity equivalents (RAE) for their
Dietary Reference Intakes, defined as follows:
Retinol activity equivalents (RAEs)
1 µg RAE = 1 µg retinol
1 µg RAE = 2 µg all-trans-β-carotene from supplements
1 µg RAE = 12 µg of all-trans-β-carotene from food
1 µg RAE = 24 µg α-carotene or β-cryptoxanthin from food
Different methods have been proposed for the analysis of carotenoids including β-
carotene. Methodology for the estimation of β- carotene from a variety of food material is
outlined below:
Principle
The sample is extracted in acetone which dissolves both the fat and water soluble
pigments. The acetone extract is then taken in petroleum ether layer. The fat soluble
carotenoid pass from acetone to the petroleum ether leaving all the rest of the pigment
pigments in acetone.
29
Equipment and glassware
Analytical balance, Spectrophotometer, Separatory funnel/ Column, Falcone tube,
Conical flask, Oakridge tube, Centrifuge, Grinder, Morter-Pestle
Reagents
Acetone, Petroleum ether, Magnesium oxide (MgO)/ Alumina (Al2O3) /Silica, Anhydrous
Sodium Sulphate
Procedure
1) Weigh 2 to 5 g of fresh sample according to their expected β-carotene content.

2) Homogenize it with equal amount of anhydrous sodium sulphate in grinder/mortar
with pestle using 10 ml acetone solvent.
3) Transfer the homogenized material in Oakridge tube and centrifuge at 5000g for 5
min and collect the supernatant.
4) Continue the extraction of the residue with 10 ml acetone till residue is colourless
(Three times).
5) Pool all the extracts and transfer it into a separatory funnel. Add 10 15 ml of
petroleum ether in separatory funnel and shake thoroughly. The yellow pigment is
transferred into petroleum ether by diluting the acetone with water containing 5%
sodium sulphate. Continues the transfer with more petroleum ether if required
(until acetone get colourless).
or
5) Load the supernatant on to a column of alumina, which has previously been
activated by keeping in oven at 50 60oC for 48 hrs. The top of the column
contains 1cm anhydrous sodium sulphate. Elute the β-carotene with petroleum
ether.
6) Note the volume of the petroleum ether and measure the absorbance at 460 nm
after the dilution with petroleum ether.
7) Evaporate the petroleum ether under rotatory vacuum distillation to concentrate it.
Make the powder using lyophilizer. The residue was kept at 20 C, reconstituted
with 5 ml petroleum ether and dilute with petroleum ether prior to UV
measurements at 460 nm.
Preparation of Standard graph

Standard β-carotene for identification was prepared in petroleum ether to obtain
1 8 μg/ml.
30
Standard β carotene
0.9
0.789
0.8 y = 0.0984x - 0.0009
R² = 0.9999
0.7
0.586
0.6
0.5
A460
0.392
0.4
0.3
0.197
0.2
0.098
0.1
0
0 1 2 3 4 5 6 7 8 9
µg/ml
Standard curve of β-carotene.

References
Ranganna, S. (1986). Handbook of analysis and quality control for fruit and
vegetable products. Tata McGraw Hill publishing company Ltd., New Delhi pp
84-88.
Karnjanawipagul, P., Nittayanuntawech, W., Rojsanga, P., and Suntornsuk L.

(2010) Analysis of β-Carotene in Carrot by Spectrophotometry. Mahidol
University Journal of Pharmaceutical Science, 37 (1-2), 8-16.
Rodriguez-Concepcion M., Stange, S. (2013) Biosynthesis of carotenoids in

carrot: An underground story comes to light. Archives of Biochemistry and
Biophysics, 539, 110–116.
Engler, M., Hammann, S., Vetter, W. (2015) Isolation of β-carotene, α-carotene

and lutein from carrots by countercurrent chromatography with the solvent system
modifier benzotrifluoride. Journal of Chromatography A, 1388, 119-125.
Ma, T., Tian C., Luo J., Sun X., , Quan, M., Zheng, C., Zhan, J. (2015) Influence
of technical processing units on the Î±-carotene, Î²-carotene and lutein contents of
carrot (Daucus carrot L.) juice. Journal of Functional Foods, 16, 104-113
31
Estimation of vitamin C content
Introduction
Vitamin C is a highly water-soluble natural acidic bioactive compound having strong

reducing properties and well known as a wonder worker because of wide range of roles
played. This essential vitamin exists in L isomeric form (L-ascorbic acid), and the mirror
image, D-isomer (i.e., D-ascorbic acid), only has about 10% of the activity of the L-
isomer. This water soluble vitamin is a strong reducing agent, which carries out its
reducing function and easily converts to its oxidized form, the L-dehydroascorbic acid,
when oxidative stress is present. Due to this characteristic, L-ascorbic acid is commonly
applied in food industry as a food additive functioning as a versatile antioxidant to protect
foods from deterioration by oxidation. Vitamin C is an essential nutrient and its deficency
cause scurvy in humans as it functions as a cofactor in several vital enzymatic reactions.
Large concentration of vitamin C can be found in fruits like oranges, grapes, lemons,
lime, papaya, strawberries and cantaloupe. Along with this it’s also present in a variety of
vegetables like tomatoes, broccoli, green and red bell peppers, raw lettuce and other leafy
green.
Principle
This method determines the vitamin C concentration in a solution by a redox titration
using an indophenol dye. Ascorbic acid which has strong reducing property reduces 2,6
dichloro indophenol (DCPIP) dye to a colourless, leuco base in acidic medium. In this
reaction ascorbic acid is oxidised to dehydro ascorbic acid while indophenol dye, which
is violet in colour, acts as oxidising agent and gets decolourised after being reduced the
end point is an appearance of faint pink colour which persists for few seconds. The
method is suitable for use with vitamin C tablets, fresh or packaged fruit juices and solid
fruits and vegetables.
DCIPIP
The compounds involved in this redoxtion are shown in the halfreactons
Ox: vitamin C red → vitamin Cox + 2 e− + 2 H+ (1)
Red: DCP ox + 2 e− + 2 H+ → DCP red (2)
Overall: vitamin Cred + DCPox → vitamin Cox + DCP red (3)
So, vitamin C is oxidized by DCP in a 2 e–/2 H+ transfer
32
Note: This method is straight forward than the alternative method using potassium
iodate, but as the potassium iodate solution is more stable than the iodine as a primary
standard, the alternative method is more reliable.
Materials
Sample containing vitamin C
Standard ascorbic acid
2,6-dichloro indophenol dye
Sodium bicarbonate
Metaphosphoric acid
Mortar and pestle
Burette and stand
100 mL or 200 mL volumetric flask
Micropipette
10 mL and 100 mL measuring cylinders
250 mL conical flasks
Whatmann No.1 filter papers
Water bath with thermostat
Centrifuge
Reagents
2,6-dichloro indophenol dye solution: Dissolve 125 mg of sodium salt of dye and 105
mg NaHCO3 in 100 ml hot distilled water (about 80°C). Cool, filter and make the volume
to 500 ml with distilled water.
Stabilizing medium: (3%w/v): Weigh 15 g of metaphosphoric acid (HPO4), stir after
adding 50 ml of distilled water and make the volume to 500 ml and keep it in cold.
33
Standard ascorbic acid solution (1 mg per ml): Dissolve 100mg of ascorbic acid in 100
ml of stabilizing medium
Methods
Extraction of ascorbic acid from sample

1. Extract approx. 10g of sample in stabilizing medium (1:5) in mortar pestle. In
case of citrus fruits (Lemon etc), juice is directly extracted by squeezer.
2. Centrifuge the homogenate. Take the supernatant and make the volume to 100 ml
with stabilizing medium.
3. Filter it, if needed, through whatman filter paper No.1 and suitable aliquot is used
for estimation of ascorbic acid.
Standardization of dye
Take 1 ml of standard ascorbic acid (1mg per ml) in 50 ml conical flask and titrate with
DCPIP dye solution to faint pink color which should persist for 15 sec. note the titre
value.
CAUTION: Iodine stains both skin and clothing so proper care is advised. If staining
does occur, alcohol may remove skin stains and cleaners are available
for fabric stains.
Estimation of ascorbic acid in sample

Take known volume (5-10 ml) of phosphoric acid extracted sample in 50 ml conical flask
and titrate with standard dye to faint pink colour which should persist for 15 sec.
CAUTION: The importance of the room temperature being constant and not too high
is because otherwise the vitamin C content may have been altered, since
vitamin C is heat labile. Moreover, if the temperature varied, the
measured results might have varied also
Calculations
X mg V2 Z
Amount of ascorbic acid (mg per/100g) = -------------------------- 100
V1 Y ml wt of the sample
Where: X = mg of standard ascorbic acid
V1 = Titre value of standard ascorbic acid against dye
V2 = Titre value of sample against dye
Y= amount of aliquot taken (ml) for estimation
Z= Total volume made up of extracted sample
NOTE: Ascorbic acid is susceptible to oxidation by atmospheric oxygen over time. For
this reason, the samples should be prepared immediately before the titrations. However,
if the samples have to be prepared several hours earlier, oxidation can be minimised by
the addition of a small amount of oxalic acid (eg 1 g oxalic acid per 100 mL of sample
solution)
34
References
Silverstein, Dr. Alvin, Virginia and Robert. (1992). Vitamins and Minerals. Brookfield,
Connecticut: The Millbrook Press.
Sullivan, Karen. (1997). Vitamin and Minerals: A Step-by-step Guide. Rockport,
Massachusetts: Element Books Limited.
Wokes F, Organ J, Duncan J, and Jacoby FC. (1943). Apparent vitamin C in foods.
Biochem J. 37(6): 695–702.
35
Estimation of bioactive compounds using HPLC
Veda Krishnan

110012, India.
Introduction
High performance liquid chromatography (HPLC) is a versatile, robust, and widely used
technique for estimating various bioactives of plant origin. This analytical technique is a
main choice for fingerprinting studies for the quality control of herbal plants, fruits etc.
The biologically active entity like anthocyanins, isoflavones and many more are often
present only as minor component in the plant extract and the resolving power of HPLC is
ideally suited for the rapid processing of such multicomponent samples on both an
analytical and preparative scale. Many bench top HPLC instruments now are modular in
design comprising a solvent delivery pump, a sample introduction device such as an auto-
sampler or manual injection valve, an analytical column, a guard column, detector and a
recorder or a printer. Chemical separations can be accomplished using HPLC by utilizing
the fact that certain compounds have different migration rates given a particular column
and mobile phase. The extent or degree of separation is mostly determined by the choice
of stationary phase and mobile phase (isocratic or gradient). There are certain critical
factors that affect quality of HPLC based separation and quantification. Some of the
parameters are briefly described below.
Sample preparation
Sample preparation is about more than just the dissolution of s solid in a liquid. Samples
may require other techniques such as filtration, extraction or derivatization as well as
accurate weighing and /or dissolution. Filtration can be performed on-line using a pre-
column filter or as the sample is introduced to the vial.
Mobile phase
Solvent preparation is very important and can save vast amounts of time spent for
troubleshooting spurious peaks, baseline noise, etc. All solvents including water should
be of highest quality (HPLC grade). Deionized water often contains trace levels of
organic compounds and so therefore is not recommended for HPLC use. All buffers
should be prepared freshly on the day required which ensures that the pH is unaffected by
prolonged storage and lack of microbial growth. Buffer reagents can contain a stabilizing
agent, for example, sodium metabisulphite. These stabilizing agents often affect the
optical and chromatographic behavior of buffer solutions, so it is often worth buying
reagents that contain no stabilizers. Solvents should be filtered through a 0.45 or 0.2 μ
filters and degassed before use in order to avoid clogging of the column. Filtered solvents
should be stored in a covered reservoir (pyrex glass) to prevent contamination with dust.
Pump plungers, seals and check valves will perform better and lifetimes will be
maximized. Highly acidic or basic solvents are not preferred unless the column has been
36
engineered to accommodate them. For isocratic and gradient elutions, miscibility of
solvents has to be taken care of.
Column
Column, known as “heart of the chromatograph” is the stationary phase that separates the
sample components of interest using various physical and chemical parameters. For
reverse phase, HPLC commonly used to separate plant products these are usually non-
polar (C8, C18, C36 etc). They are packed with small diameter porous particles (5μ size)
bonded with certain groups on their surface (like C18) which interacts with the sample
components to separate them from one another. To protect the main column, guard
columns are also available. It should be ensured that no buffers/samples are present on
the column and proper washing using miscible solvent should be carried out after each
run. After the clean-up, ensure that the test mobile phase is compatible with the last
solvent in the column.
Oven
Retention in HPLC is temperature-dependent and hence temperature control is very
important for reproducible results and oven maintains it. If temperature varies, then it is
difficult to assign “peaks” to specific compounds in the chromatogram and the peak
areas/heights may vary. Temperature fluctuations affect solubility of certain compounds
and may result in precipitation. Certain biological compounds such as enzymes or
proteins may not be stable at room temperature or higher and have to run at 4°C.
Detection
There are many detection principles used to detect the compounds eluting from an HPLC
column. The most common are UV-Vis, refractive Index and fluorescence based
detections. Compounds of plant origins are majorly detected using spectroscopy as they
have chromophores groups. Chromophores are light absorbing groups, mostly present in
phytochemicals and their behaviour is used to allow the detection of analytes. They have
one or more detection wavelengths, each of which has a molar absorptivity associated
with it. (Table.1) comprise list of chromophores generally present in phytochemicals.
Chromatophore λmax (nm)

Acetylide 175-180
Aldehyde 210
Amine 195
Azidin 190
Azo 285-400
Benzene 184,202,255
Carboxyl 200-210
Ester 205
Ether 185
Ethylene 190
Ketone 195
Naphthalene 220,275,312
37
Nitrate 270
Nitrile 160,220-230
Nitro 210
Nitroso 302
Oxime 190
Pyridine 174,195,251
Sulfone 180
Sulfoxide 210
Thioether 194,215
Thiol 195
Estimation of Anthocyanins by HPLC

Anthocyanins are types of flavonoids present in fruits and other colored plant parts like
seed coat, leaf etc. These purple compounds are known for its antioxidant, anti-cancerous
and anti-obese effects. Finger printing of anthocyanin types also very important in berries
and grapes which determine the quality of their products like wine etc. There have been
over 500 different anthocyanins identified and 23 anthocyanidins. Anthocyanins are the
glycoside (sugar-linked) versions of anthocyanidins, which are characterized by their
unique chemical structure (aromatic ring bonded to a heterocyclic ring with one oxygen
atom, creating the flavylium ion). Among the various types cyanidin, delphinidin and
pelargonidin are the most common ones found in nature, among which cyanidin-3-
glucoside (figure1) predominates.
Figure: Chemical structure of cyanidin-3-glucoside

Sample (Black Soybean seed coats)
Acetone
Water
Methanol
Acetonitrile
Trifluoroacetic acid (TFA)
2N HCl
38
0.2μ syringe filters
Hamilton syringe
Boiling water bath
Eppendorf tubes
Mobile phase [18% solvent B (0.4% TFA in acetonitrile) in solvent A (0.4% TFA in
distilled water]
Cyanidin-3-Glucoside (standard)
Procedure
Extraction
1. Wash sample (black soybean seeds) using distilled water and heat at 100°C for 2 hrs
(seed coat can be removed and stored at -80°C)
2. Grind 100 mg seed coat to fine powder using mortar and pestle pre-chilled with liquid
nitrogen.
CAUTION: Wear gloves and safety glasses to avoid cold-burn and protecting eyes
while working with liquid nitrogen.
Extract purification
3. Sample will be incubated in 1 ml acidified methanol for 48hrs for maximal recovery of
anthocyanins.
4. Centrifuge the extract at13000 rpm for 10 min at 4°C.
5. Transfer the supernatant to a fresh tube and vacuum evaporate at 30°C
6. The powder left is mixed with 200μl mobile phase and 100μl (2N HCl) for 3hrs at
98°C
7. The digested sample is filtered through 0.2μ syringe filters prior to injection into
HPLC
Analysis
HPLC instrument conditions have to be set accordingly before analysis.

 The flow rate have to be 0.8 mL/min
 The injection volume vary from 20 μL
 The column temperature has to set at 35 ± 5°C.
 Detector has to be fixed at 530 nm (λmax)
 Mobile phase: 18% solvent B (0.4% TFA in acetonitrile) in solvent A (0.4% TFA
in distilled water
CAUTION: Glass containers are not suitable for HPLC mobile phases with pH above
8.0 as metal ions will leach from containers. In such cases stainless steel
containers are recommended. Mobile phase changeover should be
gradual and intermediate polarity solvents should be introduced in
between.
Once conditions are equilibriated, 20μl of the sample is injected into HPLC column for a
run time of 15-20 min.
39
CAUTION: Always check for pressure fluctuations, If column is clogged due to bubble or
precipitation or contamination, sudden increase in back pressure can be
seen. Wash column and pump with water to prevent deposition of solid
crystalline deposits which can cause column blockages and lead to faster
wear of pump components
Standard curve
Dissolve varied concentrations of cyanidin-3-glucoside (100-1000 ppm) in 20μl acidified

methanol can manually inject into HPLC column. The height and area of peaks will be
used for plotting the standard curve. The retention time of cyanidin-3-glecoside can be
expected at 9.2 min.
NOTE: The area or height of the peaks can be plotted against concentration of standards
for standard curve. The regression coefficient has to be more than 0.99 for accurate
results.
40
Estimation of Isoflavones by HPLC
Soybeans and soy products are the main source of isoflavones, a well-studied group of
phytoestrogens with numerous biological effects. These so-called phytoestrogens due to
their weak estrogen activity have potential protective effect against some hormone related
diseases, and a number of other biological activities potential for therapeutic purposes.
The basic structure of isoflavone aglycone consists of a 3-phenylchroman skeleton that is
hydroxylated in the 4′- and 7-positions. Depending on the substituents in the carbons 5-
and 6, mainly three types of this aglycone structure, named as daidzein, genistein and
glycitein, exist in soy. Reverse phase HPLC can separate the predominant forms –
daidzein and genistein.
(A) (B)
Figure: Chemical structure of (A) Daidzein (B) Genistein

Sample (Soybean seeds)
Acetone
Water (HPLC grade)
Ethanol (HPLC grade)
0.2μ syringe filters
Boiling water bath
Hamilton syringe
Eppendorf tubes
Mobile phase [0.1% acetic acid and methanol (52:48)]
Daidzein & Genistein (standard)
Procedure
Extraction
1. Grind 125 mg seed to fine powder using mortar and pestle pre-chilled with liquid
nitrogen.
41
3. Sample will be extracted with 5 ml of 80% ethanol containing 1 ml HCl in a boiling

water bath for 2 hrs, which result in the conversion of daidzin and genistin present in
the sample in to their aglycone forms (daidzein and genistein).
4. Centrifuge the extract at13000 rpm for 10 min at 4°C.
5. The supernatant was filtered first with mira cloth and then with 5 micron filter to get a
clear extract.
6. Further filter through 0.2μ syringe filters prior to injection into HPLC.
Analysis
HPLC instrument conditions have to be set accordingly before analysis.

 The flow rate have to be 1 mL/min
 The injection volume vary from 20 μL
 The column temperature has to set at 38 ± 5°C.
 Detector has to be fixed at 255 nm (λmax)
 Mobile phase: 0.1% acetic acid and methanol (52:48)
Once conditions are equilibriated, 20μl of the sample is injected into HPLC column for a
run time of 20-30 min
Standard curve
Dissolve varied concentrations of standards (12.5-50 ppm) in mobile phase and manually
inject into HPLC column. The height and area of peaks will be used for plotting the
standard curve. The retention time of daidzein can be expected at 10.45 min and genistein
at 14.24 min.
NOTE: The area or height of the peaks can be plotted against concentration of standards
for standard curve. The regression coefficient has to be more than 0.98 for accurate
results.
42
Estimation of Proteins and Amino acids
Suresh Kumar and Sweta Kumari
110012, India.
Introduction
Proteins are not only the body building constituent of an organism, but also the catalysts/
enzymes for biochemical reactions. Plant cells differ from animal cells in that they have
rigid cell wall, and often have vacuoles that contain secondary metabolites (especially
phenolics & polyphenolics), organic acids and proteinases. When the plant tissues are
ground into fine powder, the vacuoles are ruptured releasing the contents that may
degrade, inactivate, modify and/or precipitate the proteins. Therefore, special care is
required to protect the proteins. Though proteins are extracted in the laboratories for a
variety of reasons, there are two main purposes for their extraction from plant tissues: (i)
to estimate total protein content in crude plant extract, (ii) to purify an enzyme or a
protein. The method that should be used for protein extraction would depend on the
purpose, type of the plant sample, and the enzyme or protein to be purified and estimated.
The more we know about the characteristic features of the protein/enzyme of interest, it is
easier to choose the method of extraction. In general, younger plants tissues are easier to
grind into fine powder, and often contain fewer interfering (inactivator, phenolics,
proteinases etc.) compounds. There are several biochemical parameters that must be
considered to improve stability, protection, recovery and yield of the protein(s) to be
isolated/purified. Some of the parameters are briefly described below.
Extraction buffer
As the content of vacuoles are quite acidic, sufficient buffering capacity of the medium
must be ensured to avoid drastic change in the pH from the acceptable range for the
protein of interest. Buffer maintains pH of the medium as well as ionic-strength to
stabilise the proteins/enzymes. MOPS (N-morpholino propanesulfonic acid, pH 7.0)
buffer is the most commonly used buffer for extraction of proteins; however, other
buffers including HEPES (N-2-hydroxyethylpiperazineN -2-ethanesulfonic acid) and
MES [phosphate, or 2-(N-morpholino)-ethanesulfonic acid] can also be used depending
on the purpose of extraction of proteins and the needs to avoid a particular buffer. Certain
proteins show optimal solubility at higher pH; therefore, Tris base may be included to
raise pH of the extraction buffer. Moreover, different proteins are soluble at different pH,
so different pH of the extraction buffer may be utilized to extract different set of proteins.
Volume of extraction buffer to be used per unit weight of plant sample depends on the
type of tissue and the purpose of extraction of the proteins. Generally, better extraction of
proteins is expected with a higher ratio (for example 10:1) of buffer (volume) to the plant
sample (weight). However, for extraction of a larger amount of proteins this ratio may
result in too large volume to work with the subsequent steps. If the ratio of extraction
buffer to plant tissue is not sufficient enough, this may cause change in pH of the extract
and inactivation of the extracted proteins/enzymes. Buffer may also contain 10% 20%
glycerol to ensure stability of certain unstable proteins. Some proteins (e.g. certain seed
storage proteins) require ionic interactions for solubility, so they are soluble only in the
43
presence of higher (0.1 to 1 M) salt concentrations. Moreover, the use of salt requires
caution because of its disruptive effect in the downstream processes (e.g. separation of
proteins by isoelectric focusing).
Antioxidants
Antioxidants are added in the extraction buffer to maintain reduced state of free
sulfhydryl groups of enzymes and to reduce oxidation of other components, such as
phenolics. The antioxidants which are commonly used include DTT (2 mM or more), β-
mercaptoethanol (5 10 mM), or ascorbic acid (5 10 mM). Under certain circumstances,
even stronger reducing agent (such as sodium dithionitecan) and more than one reducing
agents (e.g. DTT and β-mercaptoethanol) can also be used. Antioxidants also reduce the
activity of polyphenol oxidases which synthesize polyphenols from phenolic compounds
upon tissue disruption in the presence of oxygen.
Detergents
Detergent [e.g. Sodium Dodecyl Sulfate (SDS)] is used in the extraction buffer to
disrupt cell membranes. Under certain circumstances, they are useful for maximizing the
yield of soluble proteins, especially when low ratio of extraction buffer to tissue is used.
Other detergents [e.g. Tween 80 (0.1% 1%), Triton X-100 (0.1% 1%)] can also be used
for differential extraction. In general, initial testing is required to determine the optimal
combination and concentration of the detergents to be used.
Protective agents
Protective agents are generally required in the extraction buffer to prevent polyphenolic
compounds [e.g. tannins (water-soluble polyphenols of varying molecular weight)] to
bind and inactivate proteins. Generally, Polyethylene glycol (PEG 6000), insoluble PVPP
or soluble PVP (polyvinyl pyrrolidone) are used in most of the applications which bind
polyphenols best at the pH < 7. PVPP is used @ 0.5 5% (w/v) which requires to be
hydrated for 1 hour in the extraction buffer before use, and it can be removed from the
medium by filtration and/or centrifugation.
Proteinase inhibitors
Proteinases are released from vacuoles upon disruption of the cells; therefore, use of
proteinase inhibitors is essential for minimising the activities of proteinase and to control
degradation of proteins. Cysteine proteinases are most common in plant tissues but
metallo-, serine, and aspartic proteinases have also been reported in different tissues.
Some proteinase inhibitors are irreversible and chemically modify their targets (for
example, phenylmethylsulfonyl fluoride, PMSF), whereas others are tight-binding (for
example, bovine pancreatic trypsin inhibitor, BPTI). On the other hand, some proteinase
activity may be recovered in later steps if proteinase inhibitor is not present. Generally,
PMSF (1 mM) and BPTI (1 μM) are used against serine proteinases. Since PMSF is toxic
and rapidly hydrolyzed, it must be used carefully and should be added to the extraction
buffer just before tissue disruption. EDTA (1 10 mM) is used against metallo-
proteinases, while trans-epoxysuccinyl-L-leucyl-amido-(4-guanidino) butane, (10 μM) is
effective against cysteine proteinases. Antipain and leupeptin (10 μM) have activity
against all serine and cysteine class proteinases. Commercial proteinase inhibitors
(available in the form of tablet from Roche, or solution from Sigma) containing a cocktail
of these inhibitors can be purchased from the supplier. It must be remembered that
proteinase inhibitor tablet is slow to dissolve; therefore, it should be added to the
extraction buffer about 30 min before using the buffer for protein extraction.
44
Tissue types
The type of tissue used for protein extraction determines the method which should be
used and the yield of protein which can be obtained. In leaf, protein concentration is high
(1% 4%), while stem tissues may have lower amount of proteins. Therefore, use of
higher extraction buffer ratios will lead to very low concentration of proteins in the
extract. Seeds (usually having <10% water) have higher protein concentration, but also
often contains high amount of lipids which creates difficulty to separate them from the
extract. Therefore, tissues with high lipid content, may need to be defatted before further
purification. Fruits are often acidic, with high levels of polyphenolic compounds,
therefore require higher buffer ratio/concentration to maintain the pH of the extraction
medium. Tissue cultured cells and single-celled algae may be difficult to disrupt, hence
use of sonication, French press, or cell wall–digesting enzyme (macerozyme) may be
required.

Frozen or fresh plant tissues
Liquid nitrogen
Extraction buffer
Mortar and pestle or Tissue grinder
Miracloth or nylon mesh (20–70 μ pore size) for filtration
Refrigerated centrifuge and centrifuge tubes
Extraction buffer
0.2 M MOPS, pH 7.0
5% (w/w) PVPP (to be added into extraction buffer for hydrating about 1 hour before
use)
1% (v/v) Triton X-100 (optional)
10% (v/v) glycerol (optional)
The buffer with above components can be stored at 4 C for several weeks.
On the day of use, add the following:
2 mM Dithiothreitol (DTT)
Suitable plant proteinase inhibitor cocktail (Sigma, Roche, or equivalent) can also be
add according to the manufacturer’s instructions, or 30 min before tissue extraction.
NOTE: Use Milli-Q water or double-distilled water for the preparation of all the buffers and solutions.
Methods
Tissue disruption
1. Grind the fresh or frozen plant material to a fine powder using mortar and pestle pre-
chilled with liquid nitrogen.
2. Transfer the frozen sample powder to ice-cold extraction buffer, mix quickly and
thoroughly.
45
CAUTION: Do not add buffer to the chilled ( 196°C) mortar and pestle, as the
sample will be frozen and may take long time to thaw. Instead, add the
frozen powder to the extraction buffer and mix them quickly.
3. Filter the extract immediately through Miracloth or nylon mesh (20 70 μ) to remove
cell debris.
4. To remove insoluble materials from the filtered extract, centrifuge it at maximum
speed (12000 x g) for 10 min at 4°C.
5. Decant the supernatant carefully, avoiding pouring in of the unstable pellet.
NOTE: All operations should be carried out at 0 4°C (in a cold room or on ice). The
extraction procedure should be performed rapidly to minimize exposure of
proteins of interest to potentially damaging compounds and enzymes released
upon rupture of the cells.
Alternative Protocol
 Cut the plant material into small pieces

 Weigh 0.5 1.0 g of the plant tissues and extract with 1 ml of 0.1M phosphate
buffer (pH 7.5) using pestle and mortar.
 Centrifuge the extracted material at 10,000 x g at 4ºC for 15 min.
 Decant the supernatant and discard the pellet.
 Add equal volume of 15% TCA to the supernatant to precipitate the protein and
keep the solution overnight at room temperature
 Centrifuge the above solution at 5,000 g for 10 min.
 Decant and discard the supernatant. Dissolve the precipitate in 0.1 N NaOH
solution and make up the volume to 10 ml with 0.1 N NaOH. This solution can be
used for protein estimation by Lowry/Bradford method.
46
Estimation of Protein by Lowry Method
Lowry (1951) method of protein estimation is one of the often-cited methods. For some
time, it has been the method of choice for estimation of protein in cell fractions,
chromatography fractions, enzymatic preparations etc. The Lowry method is sensitive to
low concentration (10 g/ml) of protein and the sensitivity is moderately constant from
one protein to another. The method is based on the Biuret reaction, where Cu+, produced
by the oxidation of peptide bonds, reacts with Folin–Ciocalteu reagent (a mixture of
phosphotungstic acid and phosphomolybdic acid). The major disadvantages of the Lowry
method are the narrow pH range within which it is accurate, and the colour development
is partly dependent on the content of aromatic amino acids in the protein.
Principle
Under alkaline conditions the divalent copper ion (Cu2+) forms complex with peptide
bond during which it is reduced to a monovalent ion (Cu+). Monovalent copper ion and
the radical groups of tyrosine, tryptophan, and cysteine react with Folin’s reagent to
produce an unstable product that becomes reduced to molybdenum/tungsten blue.
Reagents
A. 2% Na2CO3 in 0.1 N NaOH
B. 1% NaK Tartrate in H2O
C. 0.5% CuSO4.5 H2O in H2O
D. Reagent I: 48 ml of A, 1 ml of B, 1 ml C
E. Reagent II- 1 part Folin-Phenol [2 N]: 1 part water
BSA Standard - 1 mg/ml
Procedure
Prepare standard (known) protein (BSA) solutions of different concentration (e.g.
200, 400, 600, 800, 1000 µg BSA per ml) in 5 test tubes and make up the volume
to 1.0 ml using distilled water.
Test tube with 1.0 ml distilled water (0 µg BSA per ml) will serve as a blank.
Add 4.5 ml of Reagent I and incubate for 10 min.
After incubation, add 0.5 ml of Reagent II and incubate for 30 min.
Measure the absorbance at 660 nm, and plot the standard curve (as shown in the
example below).
To estimate the amount of protein present in the given samples, take 100 or 200
µl of the protein extract and make up the volume to 1.0 ml using distilled water.
Follow the steps 3 5, and find out the amount of protein present in the sample(s)
using the standard curve (as explained in an examlpe below).
References
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the
Folin phenol reagent. J. Biol. Chem. 193: 265–75.
47
Estimation of Protein by Bradford Method
Bradford method of protein estimation uses a concept different from that of the Lowry’s
method. In this method, protein’s capacity to bind (quantitatively) with a dye is utilized to
measure protein concentration in a given sample. For estimation of total proteins, a dye
[e.g. Coomassie brilliant blue (CBB)] which exhibits change in its spectral properties on
binding to the proteins is utilized. At low pH, CBB in its free (unbound) form shows
absorption maxima at 465 and 650 nm, but when it binds with proteins it shows highest
absorbance at 595 nm. There are several practical advantages of using this method: (i)
reagent preparation is simple, (ii) the colour develops rapidly and is stable, (iii) the
method is sensitive enough to estimate protein concentration as low as 20 g/ml.
However, like Lowry’s method, this is also a relative method of protein estimation since
the amount of dye binding varies with the content of the basic/cationic amino acids (e.g.
arginine, tyrosine) in the protein. Moreover, many proteins do not dissolve properly in the
acidic reaction medium.
Principle
This method is based on the ability of proteins to bind with CBB (G250) that form a
complex whose extinction coefficient is much greater than that of the free dye. It is the
anionic form of the dye that binds to proteins. Binding of CBB (blue form) with proteins
causes shift in its absorption spectrum from 465 to 595 nm. The increase of absorbance at
595 nm is proportional to the amount of bound dye, and thus to the amount
(concentration) of protein present in the sample. Since the dye binds more readily to the
cationic residues (e.g. lysine and arginine), the response of the Bradford method depends
on the amino acid composition of the protein, which is a major drawback of the method.
Materials, Equipments
• Test tubes
• Measuring cylinder
• Weighing balance
• Spectrophotometer
Reagents
• Dissolve 100 mg of Coomassie-Brilliant blue (G250) in 50 ml of 95% Ethanol.
• Add 100 ml of 85% Phosphoric acid and make up the volume to 600 ml with
distilled water.
• Filter the solution and add 100 ml of glycerol, then make up the volume to 1000 ml.
• The solution can be used after 24 hours.
BSA Standard - 1 mg/ml
Procedure
Prepare different solutions of known protein concentration by taking 0.2, 0.4, 0.6,
0.8 and 1.0 ml of standard BSA solution from the stock solution into a series of test
tubes, and make up the volume to 1.0 ml with distilled water.
Use 0.2 ml of the protein sample extract in two other tubes (to conduct the
experiment in duplicate) and make up the volume to 1.0 ml with distilled water.
Use a tube with 1.0 ml of water as a blank.
48
Add 5.0 ml of Coomassie brilliant blue to each tube and mix well by vortexing.
Wait for 30 min, and take reading of the standards and the sample(s) at 595 nm.
Plot absorbance (OD) of the standards verses their protein concentrations.
From the standard curve, find out the amount of proteins in the unknown sample(s).
For example, if the OD value for a test

sample is 0.53; then from the standard
curve we can find that the protein
concentration is 800 µg per 200 μl of the
protein sample extract.
If 1.0 g of leaf tissue was extracted with
1000 µl of phosphate buffer, and
200 µl of the extract was used to
estimate the proteins, the protein
concentration in the leaf sample would
be 800 µg x 5 = 4.0 mg/g leaf tissues.
NOTE: The OD value of test sample should be in the range of 0.2 0.8, because
spectrophotometer operates best (gives maximum accuracy) in the range of 0.2 - 0.8 OD.
If you find the OD value of your sample more than 0.8, make appropriate dilution and
take the reading again. But do not forget to use the dilution factor in calculations. For
example, if you have made double dilution (2x dilution) of the sample in the above
mentioned protein estimation, the protein concentration would be 800 x 2 = 1600 µg x 5 = 8.0
mg/g of the leaf tissues.
References
Bradford MM (1976) Rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:
248–254.
49
Estimation of Amino Acids
Amino acids are colourless ionic compounds that form the building blocks of proteins. There
are 20 different standard amino acids found in proteins. Amino acids are comprised of a
carboxyl group and an amino group attached to the same carbon atom (the α-carbon).
Amino acids vary in their size, structure, electric charge and solubility in water because
of the variation in their side chain (R group). Apart from being bound in proteins, amino acids
also exist in the free form in many tissues and are known as free amino acids. They are mostly
water soluble in nature. Often in plants, during abiotic and biotic stresses, there is a
change in free amino acid composition. Hence, measurement of the free amino acids
provides the biochemical and health status of the plant. When amino acids with a free α-
amino group are treated with an excess of ninhydrin, they yield a purple coloured
product. Under appropriate conditions, the intensity of the colour produced is
proportional to the concentration of amino acids.
It is the intensity of this purple colour which is measured spectrophotometrically

to estimate amino acids. Ninhydrin reacts with a free α-amino group (NH2 C COOH)
which is present in all amino acids, proteins or peptides. Theoretically only amino acids
can produce colour with the ninhydrin reagent. However, one should check the possible
interference from peptides and proteins by performing blank tests. For example, by
simply adding ninhydrin reagent to a solution of proteins only and watching if there
is any colour development. There is no excuse for failing to perform such test when the
sample mixture contains both proteins and amino acids. Imino acids like proline, the
guanidino group of arginine, the amide groups of asparagine, the indole ring of
tryptophan, the sulfhydryl (SH) group of cysteine, amino groups of cytosine and guanine,
and cyanide ions also react with ninhydrin to form various chromophores that can be
analyzed. Several other reagents are also available which can react with alpha amino
group to form coloured or fluorescent derivatives. These include fluorescamine, dansyl
chloride, dabsyl chloride, etc., used in the detection of trace amounts of amino acids at
the nano-gram level.
Estimation of Free Amino acids

Principle
Ninhydrin, a powerful oxidizing agent, decarboxylates the α-amino acids and yields an
intensely coloured bluish purple product which is colorimetrically measured at 570 nm.
Ninhydrin + alpha-amino acid Hydrindantin + Decarboxylated Amino acid

+ Carbondioxide + Ammonia
Hydrindantin + Ninhydrin +Ammonia Purple coloured product + Water
Reagents
• Ninhydrin: dissolve 0.8 g Stannous chloride (SnCl2.2H2O) in 500 ml of 0.2 M Citrate buffer (pH
5.0), add this solution to 20 g of Ninhydrin in 500 ml of Methyl cellosolve (2 methoxy-ethanol)
• 0.2M Citrate Buffer (pH 5.0)
• Diluent Solvent: Mix equal volumes of water and n-propanol.
50
Procedure
Extraction of Amino Acids

Weigh 500 mg of plant tissues and grind it in a pestle and mortar with a small quantity of acid-washed
sand (if the tissue is very tough, use boiling 80% ethanol for extraction). To this, add 5 – 10 ml of
80% ethanol, centrifuge it at 8000 x g and collect the supernatant. Repeat the extraction twice
with the residual sample and pool all the supernatants. Reduce the volume, if necessary, by
evaporation and use the extract for quantitative estimation of total free amino acids.
Estimation
1. Take 100 μl of the extract, add 1.0 ml of Ninhydrin.
2. Make up the volume to 2.0 ml with distilled water.
3. Heat the tube in a boiling water bath for 20 min.
4. Add 5.0 ml of the Diluents solvent and mix well the contents.
5. After 15min, measure OD (intensity of the purple colour) against a reagent
blank using a colorimeter/spectrophotometer at 570 nm. The colour is stable for 1 hour.
6. Prepare the reagent blank as mentioned above by taking 100 μl of 780% ethanol instead of the
sample extract.
Standard curve
Dissolve 50 mg leucine in 50 ml of distilled water in a volumetric flask. Take 10 ml of this stock solution
and dilute to 100 ml in another flask to prepare working standard solution. A series of volume from 0.1 to
1.0 ml of this standard solution gives a concentration range of 10 µg to 100 µg leucine (free amino acid).
Follow the procedure of colour development (as mentioned above for the sample) and
take absorbance at 570 nm. Draw a standard curve using absorbance versus concentration
of the amino acid. Using the standard curve, you can find out the concentration of total
free amino acids in the sample, which can be expressed as percentage equivalent of
leucine.
51
Estimation of Proline
Proline (Pro, P) is a basic amino acid found in high percentage in proteins. Free proline
has been proposed to act as an important osmolyte and osmoprotective compound, acting
as molecular chaperon in osmotic adjustment and protection of cellular structures,
proteins and cellular membranes during abiotic stresses. Accumulation of proline has
been associated with water deficit stress, salinity and other abiotic stresses in plants. It
accumulates under the stress in both leaf and root tissues and protects the plant from the
stress. Many researchers have reported several-fold increase in proline content under
physiological and pathological stresses; hence, analysis of proline in plants has become
routine in pathology, biochemistry and physiology laboratories.
Principle
By selective extraction with aqueous sulphosalicylic acid proteins are precipitated as a

complex. Other interfering materials are also removed along with the protein-
sulphosalicylic acid complex. The extracted proline is then made to react with ninhydrin
in acidic condition (pH 1.0) to form chromophore (red colour) and the intensity of the
colour is measured at 520 nm.
Reagents
Acid-ninhydrin: Prepared by warming 1.25 g Ninhydrin in 30 ml glacial acetic
acid and 20 ml 6 M phosphoric acid, with agitation, until dissolved completely.
Store the acid-ninhydrin at 4°C, the reagent is stable at this temperature for 24
hours.
Extraction buffer: 3% (w/v) aqueous Sulfosalicylic acid.
Glacial acetic acid
Toluene
Proline (to prepare standard curve).
Procedure
1. The plant tissue is homogenized in 3% aqueous sulphosalicylic acid, and
centrifuged to remove the residue is by centrifugation at 12000 x g for 10 min.
2. 1.0 ml of the supernatant is mixed with 1.0 ml acid-ninhdrin and 1.0 ml of glacial
acetic acid in a test tube, the mixture is boiled in a water bath (100°C) for 1 hour.
3. The reaction is terminated by putting the tube in an ice bath.
4. The reaction mixture is extracted by adding 2.0 ml toluene, mixing vigorously and
keeping at room temperature for 30 min until two phases are separated.
5. The chromophore-containing toluene (1.0 ml, upper phase) is warmed to room
temperature and its OD is measured at 520 nm using toluene as a blank.
6. The proline concentration in the test sample is determined from a standard curve
prepared using D-Proline.
Calculations
The proline content is expressed on fresh-weight basis using the following formula:
where 115.5 is the molecular weight of proline.
52
CAUTION: Ninhydrin is irritant to skin and respiratory system; therefore, safety
measures require adequate precautions, in particular wearing of gloves.
References
Bates LS (1973) Rapid determination of free proline for water-stress studies. Plant and
Soil 39: 205-207.
53
Estimation of Lysine content
Definition
Lysine content in this method refers to the total content of the amino acid lysine present
in the sample, which is expressed in percentage of the sample weight
Introduction
The importance of “available” lysine (lysine residue in which the €-amino group is free)
in protein chemistry and nutrition is well recognized. A lysine misbalanced diet results in
many severe diseases. So it is often used as a fortifier in order to improve the nutritional
values and to cure possible deficiency diseases of this particular amino acid. Owing to
this, lysine content in foods can be used as an index of nutritional quality. The accurate
and rapid estimation of lysine is of great interest because of its importance in human
ailment.
Recently, several analytical methods have been proposed for the quantitative analysis
of lysine content, including flow injection chemiluminescence, amperometric biosensor,
chromatography, and spectroscopy. The applications of the above quantitative methods
are limited by complicated pre-concentration, time-consuming steps, and requirement of
costly instruments. Therefore, the search for a rapid and simple method for the estimation
of lysine content is still desirable. A limiting factor in the determination of lysine in
grains has been a lack of reliable/reproducible technique. After reviewing the advantages
and disadvantages of various colorimetric methods, the Division of Biochemistry, ICAR-
I.A.R.I., New Delhi adopted a modified and standardized method of Tsai et al (1972) to
estimate lysine content in grains.
Principle
This method utilizes the compound 2-chloro-3, 5-dinitropyridine (CDNP) which reacts
with amino acids and peptides of low molecular weight that are present in the hydrolyzed
protein. The €-dinitropiridile (€-DNP) lysine thus formed is water soluble but insoluble in
ethyl acetate, which reacts with the €-amino group of lysine after having blocked with
copper, the α- groups of the amino that other compounds formed in the reaction be
eliminated with that solvent, along with the excess 2-chloro-3,5-dinitropyridine after
acidification with HCl. Absorbance of the aqueous solution of €-dinitropyridile lysine is
measured using a spectrophotometer at 400 nm.
Materials Powdered rice grain

Equipments
Analytical balance, Incubator oven, Shaker, Vortex mixer, Centrifuge, pH meter,
Colorimeter, Microcentrifuge tubes (2 ml), Calibrated Falcone tubes, Oakridge tubes,
Glassware (pipets, beakers, etc.).
Chemicals/Reagent (0) opper phosphate reagent: This reagent is a suspension of

copper phosphate in borate buffer.
 Borate buffer (0.05 M, pH 9.0)
 Copper phosphate suspension: prepared by dissolving 1.4 g CuCl2.2H20 in 50 ml
distilled water (Reagent A) and 6.8 g Na3P04. 12H20 in 100 ml distilled water
54
(Reagent B). Mix reagents A and B; centrifuge at 2,000 rpm for 5 minutes and
discard the supernatant. Resuspend the precipitate in 15 ml borate buffer (repeat it
4 times) and then suspend the final precipitate in 40 ml borate buffer. This reagent
can be used for two weeks after preparation.
 Pyridine reagent (3%): Dissolve 30 mg 2-chloro-3, 5-dinitropyridine in 1 ml
methanol. (prepare just prior to the use)
 Carbonate buffer (0.5 M, pH 9.0)
 1.0 N HCI solution
 Papain (4 mg/ml for sample and 5 mg/ml for standard): Prepared in 0.05 M
phosphate buffer, pH 7.4 or 0.01 N sodium acetate pH 7.0.
Procedure
Weigh 100 mg finely ground, defatted rice powder and add 5 ml papain solution.
Make sure that the sample is thoroughly wetted; shake tubes at least twice in the
first hour of incubation. Include blank of just papain solution.
Incubate at 63±2°C for 16 hours. Shake, and cool down to room temperature and
then centrifuge at 10,000 rpm for 5 minutes.
Pipette 1.0 ml aliquots of the supernatant into microcentrifuge tubes and add
0.5 ml carbonate buffer (0.5 M, pH 9.0) and 0. 5 ml copper phosphate reagent.
Shake the mixture for 5 minutes and centrifuge at 10,000 rpm for 10 minutes
Pipette 1.0 ml aliquots of the supernatant into large Falcon tubes and add 0.1 ml
2-chloro-3, 5-dinitropyridine. Shake well.
Allow the mixture to stand at room temperature for 2 hours, shaking at every 30
minutes interval.
Add 4 ml 1.0 N HCI to each test tube and shake well.
Add 5 ml ethyl acetate and mix well, invert the capped tubes at least ten times;
extract the upper phase. This step must be performed three times
Transfer the aqueous phase to calibrated tubes and take the absorbance at 400
nm, adjusted with the blanks.
Calibration Curve
Prepare a standard curve with a range of 0 to 800 μg lysine per ml.
Stock solution of lysine: 16 mg lysine-monohydrochloride in 2.0 ml carbonate buffer.
Dilute the stock solution of lysine to 0, 500, 1000, 2000, 3000, 4000 μg lysine per ml.
To 1.0 ml of each of the solutions, add 4 ml papain solution (5 mg papain per ml
phosphate buffer). Pipet 1 ml of each solution into centrifuge tubes and add 0.5 ml
carbonate buffer (0.5 M, pH 9.0) and 0.5 ml copper phosphate suspension. Continue
with step 4 of Procedure.
Calculations
% Lysine = OD 400 /Slope (OD/µg /ml) x Hydrolysis volume (ml)/sample wt. (µg) x
100
Precautions
Borate buffer, carbonate buffer, phosphate buffer should be kept at 4 °C. It will
be stable for several weeks.
Papain should be prepared, every time you will use it. Ensure that the
55
phosphates buffer is at room temperature and papain powder should well
dissolve.
Copper phosphate reagent should be stored at 4 °C and it will be stable for one
weeks. Prepare the pyridine reagent daily. Protect it from light and oxygen. Be
sure that dinitropyridine is completely dissolved.
Lysine solution should be prepared weekly and vortex thoroughly before you
prepare dilutions for standard curve.
For better results sample should be defatted in hexane.
References
Tsai, C.Y., L.W. Hansel and O.E. Nelson. A (1972) Colorimetric method of screening
maize seeds for lysine content. Cereal Chemistry, 49: 572-579.
Villegas, E., and E.T. Mertz. (1970) Screening techniques used at CIMMYT for protein
quality maize. Technical Bulletin No. 20. International Maize and Wheat Improvement
Center, Mexico.
Galicia, L., Nurit, E., Rosales, A. and Palacios-Rojas N. (2008) Maize nutrition quality
and plant tissue analysis laboratory. Laboratory Protocols, Mexioc, D.F.: CIMMYT.
ISBN: 978-970-648-169-6
56
Protein Characterization Using Different Gel-Based
Proteomic Tools
Ranjeet R. Kumar, Suneha Goswami, and Shelly Praveen

110012, India.
Introduction
A proteome is a set of proteins produced in an organism, system, or biological context.
The proteome is not constant; it differs from cell to cell and changes over time. To some
degree, the proteome reflects the underlying transcriptome. However, protein activity
(often assessed by the reaction rate of the processes in which the protein is involved) is
also modulated by many factors in addition to the expression level of the relevant gene.
Proteomics is used to investigate the time and duration of proteins expression, the rate of
protein synthesis and degradation, localization of proteins and their modifications, etc.
Proteome has evolved with the advent in technology and most of the tools used for the
protein characterization have been classified into two different groups – Gel-based
proteomics and Gel-free proteomics. The techniques like Native-PAGE, SDS-PAGE,
Two-dimensional electrophoresis (2-DE), Two-dimensional differential gel
electrophoresis (2-DIGE) are basically used for the gel-based proteomic characterization,
whereas nLC-MS, nLCMS2, LTQ-Orbitrap and recently developed technique i.e.
Isobaric Tag for Relative and Absolute Quantitation of Protein (iTRAQ) has been
included under gel-free proteomic tools or Fourth generation proteomic tools.
Blue native-PAGE is technique used for characterizing the biological membranes,

complex proteins, to determine native proteins masses and oligomeric states, etc. In this
method CBB dye is used to provide the charges required for the electrophoretic
separation of the proteins. Clear native-PAGE is used for the separation of acidic water-
soluble and membrane proteins in a gradient gel. The electrophoretic mobility of the
proteins in CN-PAGE is based on the intrinsic charge of the proteins. Quantitative Native
PAGE is frequently used for the separation of metalloproteins or enzymes. The proteins
are continuously separated using eluents and collected in a fractionator. The whole
process is repeated twice or thrice in order to identify the metal cofactor.
57
Native Polyacrylamide Gel Electrophoresis
(Native-PAGE)
Polyacrylamide gel electrophoresis (PAGE) offers high resolution of low-molecular-
weight nucleic acids and proteins. In particular, small DNA fragments (<500 bp) that are
poorly resolved by ordinary agarose gels are easily separated on polyacrylamide gels.
Depending on the pore size of the gel (3.5% to 20% polyacrylamide), a separation from
10 to 1000 bp can be achieved.
A polyacrylamide gel is formed by the polymerization of acrylamide monomers

into long chains, which are further covalently attached by a cross-linking agent, most
commonly N,N'-methylene-bis-acrylamide. Polymerization of a polyacrylamide gel is
initiated by free radicals provided by ammonium persulfate and stabilized by TEMED.
The reaction takes about 10 to 20 min to go to completion, but the reaction rate can be
varied by adjusting the TEMED and ammonium persulfate concentrations. The pore size
of a polyacrylamide gel is determined by the total percentage of acrylamide (the sum of
the weights of the acrylamide monomer and cross-linker). Historically, this has been
expressed as %T. As the %T increases, the pore size decreases. The migration distance
(D) of double stranded DNA through a non-denaturing gel is inversely proportional to the
log of its molecular weight (DÅ –log (MW)). Also, the base composition of a sequence
affects its electrophoretic mobility and may cause aberrant migration.
Under native PAGE conditions, polypeptides retain their higher-order structure

and often retain enzymatic activity and interaction with other polypeptides. The migration
of proteins depends on many factors, including size, shape, and native charge. The
resolution of non-denaturing electrophoresis is generally not as high as that of SDS-
PAGE, but the technique is useful when the native structure or enzymatic activity of a
protein must be assayed following electrophoresis. One straightforward approach to
native electrophoresis is to omit the SDS and reducing agent (DTT) from the standard
Laemmli’s SDS protocol.
Reagents/instruments
Stock solutions
Acrylamide solution (30% acrylamide, 0.8% bis-acrylamide, 200 mL)
Final concentration Amount

Acrylamide (F.W. 71.08) 30% 60 g
bis-acrylamide (F.W. 154.17) 0.8% 1.6 g
ddH2O to 200 mL
Note: This will make a 30.8% T 2.7% C bis solution. Store up to 3 month at 4 °C in the dark.
4x Resolving gel buffer (1.5 M Tris-Cl, pH 8.8, 200 mL)

Tris (F.W. 121.1) 1.5 M 36.3 g
ddH2O 150 mL
HCl To pH 8.8
ddH2O To 200 mL
Note: Store up to 3 month at 4 °C.
58
4x Stacking gel buffer (0.5 M Tris-Cl, pH 6.8, 50 mL)

Tris (F.W. 121.1) 0.5 M 3g
ddH2O 40 mL
HCl To pH 6.8
ddH2O To 50 mL
Note: Store up to 3 month at 4 °C.
10% Ammonium persulphate (Initiator)

Ammonium persulphate (FW 10% 0.1 g
228.2)
ddH2O 1 mL
Note: Prepare just prior to use; do not store.
Resolving Gel Overlay (0.375 M Tris-Cl, pH 8.8, 100 mL)

4x resolving gel buffer 1.5 M 25 mL
ddH2O 100 mL
2x Sample Buffer (0.125 M Tris-Cl, 20% v/v glycerol, 0.02% bromophenol

blue, pH 6.8, 10 mL)

4x stacking gel buffer 0.125 M 2.5 mL
Glycerol 20% 2.0 mL
Bromophenol blue 0.02% 2.0 mg
ddH2O To 10 mL
Note: Store in 0.5 mL aliquots at -20°C for up to 6 month.
Tank Buffer (0.025 M Tris, 0.192 M glycine, pH 8.3, 10 Lit.)

Tris (F.W. 121.1) 0.025 M 30.28 g
Glycine (F.W. 75.07) 0.192 M 144.13 g
ddH2O To 10 Lit.
Note: This solution can be made up directly in large reagent bottles, because it is not necessary
to check the pH. Store at room temperature for up to 1 month.
Water saturated n-Butanol
Amount
n-Butanol 50 mL
ddH2O 5 mL
Note: Combine in a bottle and shake. Allow phases to separate. Use the top phase to
overlay gels. Store at room temperature indefinitely.
59
Additional reagents
Protein markers
Tetramethylethylenediamine (TEMED)
Equipment’s
Vertical slab gel unit with casting stand
1.5- or 0.75-mm-thick combs and spacers
Glass plates
Pipettor capable of pipetting 1–20 μL with long, narrow “gel-loading” pipette tips
Water aspirator or vacuum pump with trap
Boiling-water bath or 100 ºC temperature block
Power Supply (300 V)
Magnetic stirrer
Pipettes/graduated cylinders
Procedure
Prepare the resolving gel

1. Assemble the vertical slab gel unit in the dual-gel casting stand.
2. In a 125 mL side-arm vacuum flask, mix either 40 mL (0.75 mm) or 80 mL (1.5
mm) of resolving gel solution, leaving out the ammonium persulphate and the
TEMED.
3. Stopper the flask and apply a water vacuum for several minutes to deaerate the
solution while swirling the flask.
4. Add the TEMED and ammonium persulphate and gently swirl the flask to mix,
being careful not to generate bubbles.
5. Pipette the solution down the spacer into each sandwich to a level about 4 cm
from the top. A 25 mL pipette works well for this step.
6. Fill a transfer pipette or syringe with water-saturated n-butanol (or water or
resolving gel overlay). Position the pipette or needle at about a 45° angle with the
point at the top of the acrylamide next to a spacer. Gently apply approximately 0.3
mL of n-butanol. Repeat on the other side of the slab next to the other spacer. The
n-butanol will layer evenly across the entire surface after a minute or two. Repeat
this process to overlay the second slab. A very sharp liquid-gel interface will be
visible when the gel has polymerized. This should be visible within 10–20 min.
The gel should be fully polymerized in about 1 h.
7. After polymerization, tilt the casting stand to pour off the overlay and rinse the
surfaces of the gels twice with resolving gel overlay.
8. Gels may be stored at this point. The stacking gel is cast later. Remove the n-
butanol and add approximately 10 mL of 1x resolving gel overlay solution to the
top of each sandwich, seal with plastic wrap, and store flat at 4°C. Or store gels
submerged flat in 1x resolving gel overlay at 4°C for up to 1 week.
9. Add approximately 1 mL of resolving gel overlay to each gel and allow the gels
to sit while preparing the stacking gel.
60
Prepare the stacking gel
1. In a 50 mL side-arm vacuum flask, mix 10 mL (for 0.75-mm-thick gels) or 20 mL
(for 1.5-mm thick gels) of stacking gel solution, leaving out the ammonium
persulphate and the TEMED.
2. Deaerate as in step 3.
3. Add the ammonium persulphate and TEMED. Gently swirl the flask to mix.
4. Pour off resolving gel overlay from the gel. Remove all liquid before proceeding.
5. Fill each sandwich with stacking gel solution and insert a comb into each
sandwich, taking care not to trap any bubbles below the teeth of the comb.
6. Oxygen will inhibit polymerization, and bubbles will cause a local distortion in
the gel surface at the bottom of the wells.
7. Allow the gel to sit for at least 30 min.
8. A very sharp interface will be visible when the gel has polymerized. This should
be visible within 10–20 min. The gel should be fully polymerized after 1 h. In
general, stacking gels should be cast just before use. The complete gel can be
stored overnight at 4 °C, however, with little effect on resolution, if covered and
the comb left in place.
Prepare the sample
1. Combine equal volumes of protein sample and 2x sample buffer in a tube and mix
it properly.
If the gels will be stained with Coomassie Blue, use a starting sample protein concentration of 10–
20 mg/mL (i.e. 10–20 μg/μL). This will be diluted by the 2x sample buffer to give 5–10 μg/μL. For
complex mixtures (e.g. cell lysates), 50 μg of protein (5–10 μL of treated sample) per lane is
recommended. For highly purified proteins, 0.5–5 μg per lane is usually adequate. Silver staining
requires 10- to 100-fold less protein per lane.
2. The treated sample can be stored at 20°C for 6 month for future runs.
Loading the gel
3. Slowly remove the combs from the gels, raising the comb up to avoid disturbing
the well dividers.
4. Rinse each well with tank buffer, invert the casting stand to drain the wells, and
return the stand to an upright position.
5. Fill each well with tank buffer.
6. Using a pipettor with a long, thin tip, gently load 5–10 μL of sample beneath the
buffer in each well. Load every well with the same volume of sample. If the well
is not needed, load with 1x sample buffer containing standard protein or no
sample.
This procedure ensures that each well behaves the same during separation. If an adjacent well is left
empty, the adjoining samples will tend to spread laterally during electrophoresis. When adding the
sample, be careful to maintain a sharp interface between the sample and the tank buffer. Adding the
sample too fast or erratically will lead to swirling and a diffuse loading zone. This will cause a loss
of band sharpness. Alternatively, the sample can be added and then overlaid with tank buffer. This is
more time-consuming but, when performed carefully, minimizes contamination between wells and is
particularly useful with radiolabelled samples.
61
7. If protein molecular weight standards are used, load one or two wells with 5–10
μL of the standard mixture. If the gel is to be stained with Coomassie Blue, this
volume should contain 0.2–1 μg of each standard component. If the gel is to be
silver stained, use 10–50 ng of each component.
Run the gel
8. Fill the lower buffer chamber with 4 lit of tank buffer. Carefully fill the upper
buffer chamber with tank buffer. Do not pour buffer into the sample wells,
because it will wash the sample out.
9. Put the lid on the gel unit and connect it to the Power Supply. Turn on the power
supply and adjust the voltage to 300 (so voltage is not limiting).
10. Adjust the current to 30 mA per 1.5-mm-thick gel and 15 mA per 0.75-mm-thick
gel. Start the electrophoresis. The voltage should start at about 70–80 V, but will
increase during the run. Keep a record of the voltage and current readings so that
future runs can be compared and current leaks or incorrectly made buffers can be
detected.
Under these conditions, the gel will take approximately 3–4 h to run. If it is more convenient to
run the gel for a longer period (e.g. 8 h), reduce the current to half—15 mA per 1.5-mm-thick gel;
reduce the current to 7 mA per 1.5-mm-thick gel for 16 h (e.g. overnight).
11. When the tracking dye reaches the bottom of the gel, turn the power supply off
and disconnect the power cables.
Gel Staining/destaining
12. Remove the gel from the cassette and keep it in staining solution (0.1%
Coomassie Brilliant Blue G250 in 50% methanol, 10% glacial acetic acid) for 2-3
hrs on shaker.
13. Transfer the stained gel to destaining solution (10% Methanol and 70% glacial
acetic acid) for 1-2 hrs on shaker.
14. Gel can also be used for In-gel activity assay of specific enzymes based on the
specific protocols.
References
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of
bacteriophage T4. Nature 227: 680–685.
Ornstein L (1964) Disc electrophoresis-I: Background and theory. Ann NY Acad Sci
121: 321–349.
62
Blue-Native Polyacrylamide Gel Electrophoresis
BN-PAGE was developed for the separation of mitochondrial membrane proteins and
complexes in the mass range of 10 kDa to 10 MDa. It is used for the one-step isolation of
microgram amounts of membrane protein complexes from biological membranes and total
cell and tissue homogenates, to determine native masses and oligomeric states, to
determine the stoichiometry of a multiprotein complex by an antibody-based gel-shift
method, to identify physiological protein–protein interactions, in-gel activity assays, to
isolate supramolecular physiological protein assemblies, and many more tasks.
Non-ionic detergents are used for solubilisation of biological membranes. The
choice of a specific non-ionic detergent depends on the detergent stability of the protein
complexes of interest. One of the mildest detergents is digitonin. It has been used to
isolate supramolecular associations of multiprotein complexes, thus identifying
physiological protein–protein interactions without using chemical crosslinking. Dodecyl-
maltoside has stronger delipidating properties compared to digitonin. It is a mild neutral
detergent that is suited for isolation of membrane proteins and individual complexes but
commonly dissociates labile hydrophobic interactions. Triton X-100 shows intermediate
behaviour with mitochondrial protein complexes.
Following solubilisation of biological membranes and centrifugation, the anionic
dye Coomassie blue G-250 is added to the supernatant. This dye is sufficiently soluble in
water, but it can also bind to membrane proteins because of its hydrophobic properties.
Binding a large number of dye molecules imposes a charge shift on the proteins that causes
even basic proteins to migrate to the anode at pH 7.5 during BN-PAGE. However, proteins
are not separated according to the charge/mass ratio but according to size in acrylamide
gradient gels. Protein migration gradually decelerates with running distance and with
decreasing pore size of the gradient gel. Individual proteins must stop almost completely
when they approach their size-dependent specific pore-size limit.
Because negatively charged protein surfaces repel each other, the tendency of
membrane proteins to aggregate is considerably reduced. Furthermore, membrane surface
areas lose their hydrophobic character upon binding the dye, which converts membrane
proteins into water-soluble proteins. This means that no detergent is required in the BN
gels once Coomassie dye has occupied protein surfaces. Therefore, the risk of denaturation
of detergent-sensitive proteins is minimized during BN-PAGE.
Native proteins and complexes migrate as blue bands through BN gels. This
facilitates excision of specific bands and recovery of blue stained native proteins by
electroelution. The ease of recovery is especially advantageous when proteomic work
that does not demand homogeneously dissolved enzymes is to follow34,35. BN gels
have also been used to isolate native proteins for 2D crystallization and electron
microscopic analyses26.
BN-PAGE is compatible with in-gel activity staining procedures and in-gel
detection of fluorescent labels like the CyDye fluorophors. However, interference of
Coomassie blue with activity measurements and fluorescent detection in general cannot
be excluded. In these cases, a ‘colorless native’ (CN)-PAGE system might be used instead
of BN-PAGE. In its present form, the resolution capacity of CN-PAGE, with some
exceptions, is low compared to BN-PAGE, and no suitable protocols for electroelution
and electroblotting of proteins from CN gels are available so far.
63
Reagents
1 M imidazole
1 M imidazole/HCl, pH 7.0
20% dodecyl-b-maltoside (w/v)
20% Triton X-100 (w/v)
20% digitonin (w/v)
1 M tricine
Glycerol
1 M 6-aminohexanoic acid
Acrylamide (twice-crystallized)
Bis-acrylamide (twice-crystallized)
5% (w/v) stock of Coomassie blue G-250
Ponceau S/glycerol stock solution (0.1% Ponceau S, 50% glycerol)
Tris-base, 50 mM DTT, 0.1 mM PMSF
Mercaptoethanol (for tricine-SDS-PAGE)
Equipment
Vertical electrophoresis apparatus without special cooling for BN and 2D SDS

gels.
Power supply (600 V, 500 mA) for BN-PAGE.
Table 1. Electrode and gel buffers for BN-PAGE.
Cathode Buffer Cathode Buffer Anode Buffer Gel Buffer

B A (3x)
Tricine (mM) 50 50 - -
Imidazole (mM) 7.5 7.5 25 75
Coomassie Blue G- 0.02 - - -
250 (%)
0.002 - - -
pH 7.0 7.0 7.0 7.0
Solubilisation buffers for BN-PAGE

The solubilisation buffers for BN-PAGE should be prepared as described in Table 2. But
since Bis-Tris disturbs almost all protein determination assays, it is replaced here by
imidazole buffer. Solubilisation buffer A is commonly used to solubilize mitochondria
and bacterial membranes. Higher salt concentrations should be avoided, since high salt
can lead to considerable stacking of proteins in the sample wells of BN-PAGE, and
highly concentrated membrane proteins tend to aggregate. If especially low ionic strength
conditions are desired, 500 mM 6-aminohexanoic acid in buffer B can be used instead of
50 mM sodium chloride. Low concentrations of 6-aminohexanoic acid (2 mM) and
EDTA (1 mM) are commonly added to all buffers for protease inhibition.
64
Table 2. Solubilisation buffer for BN-PAGE
Solubilisation buffer A Solubilisation buffer B
Sodium chloride (mM) 50 -
Imidazole/HCl (mM) 50 50
6-Aminohexanoic acid 2 500
(mM)
EDTA (mM) 1 1
pH 7.0 7.0
Selecting and casting gel

Select and cast the appropriate gel type for BN-PAGE. Common gel types are acrylamide
gradient gels, although, very rarely, non-gradient uniform acrylamide gels have been used
for BN-PAGE.
Uniform acrylamide gels can be optimal for separation of two protein complexes with similar masses, but
several attempts may be required to find the special acrylamide concentration for the specific narrow mass
range. First, cast gradient separation gels in the cold (4–7ºC) using a gradient mixer. The amount of each
reagent to use to compose one gel (0.16 x 14 x14 cm) is given in Table 3. Using a long cannula that
reaches to the bottom of the gel, add 1 ml of water to the bottom, and then pump in the acrylamide solution
beginning with the low acrylamide solution that is continuously underlain with solutions of increasing
acrylamide concentration and density. Alternatively, if the gel-casting device includes a hole in the spacers
between the two glass plates, the gradient gel also can be pumped from the bottom. Using a larger volume
of the 4% acrylamide solution than of the 13% acrylamide solution containing glycerol assures that the two
solutions initially are not mixed when the connecting tube is opened, and a linear gradient is obtained. 4-
13% acrylamide gradient gels cover the protein mass range 10 kDa to 3 MDa. However, proteins smaller
than 100 kDa are not well resolved. 3-13% acrylamide gradient gels extend the mass range up to 10 MDa.
Table 3. Composition of a sample gel and 4% and 13% acrylamide mixtures to prepare
an acrylamide gradient gel 10% aqueous ammonium persulfate solution must be made up
fresh for use.
Sample Gel Gradient Separation Gel
3.5% Acrylamide 4% Acrylamide 13% Acrylamide
AB-3 mix 0.44 mL 1.5 mL 3.9 mL
Gel buffer 3x 2 mL 6 mL 5 mL
Glycerol - - 3g
Water 3.4 mL 10.4 mL 3 mL
Total Volume 6 mL 18 mL 15 mL
10% APS 50 mL 100 mL 75 mL
TEMED 5 mL 10 mL 7.5 mL
Transfer the ‘gel’ to room temperature for polymerization. Polymerization takes

approximately 30 min.
Remove water from the top of the gradient gel after polymerization and cast the
sample gel at room temperature. Add the appropriate sample gel comb. Gels are used
either as ‘preparative gels’ using the total 14-cm gel width for sample application or
as ‘analytical gels’ containing 0.5-cm or 1.0-cm sample wells.
Leave the gel for approximately 15 min to polymerize.
Remove the sample gel comb and overlay the gel with gel buffer (1x).
Suspend biological membranes in buffers containing carbohydrate or glycerol (e.g.,
65
250 mM sucrose, or 400 mM sorbitol, or 10% glycerol) to avoid dissociation of
multiprotein complexes upon freezing.
Shock-freeze aliquots in liquid nitrogen and store at –80ºC. Ideally, the salt
concentration in these membrane suspensions should be low (0–50 mM NaCl).
Avoid potassium and divalent cations because these ions precipitate Coomassie dye
and proteins together with the dye. It is preferable to use 50 mM imidazole/HCl, pH
7.0. Other buffers, e.g., sodium phosphate or sodium-MOPS, are tolerated at 5–20
mM concentrations.
Use samples with low ionic strength for separation by BN-PAGE (ideally in 50 mM
NaCl, 50 mM imidazole/HCl, pH 7) to avoid protein aggregation in the sample gel
during electrophoresis. Use dialysis or other desalting techniques (e.g., centrifuge
filter units with appropriate cut-off limit, especially for high salt fractions from ion
exchangers).
Add 5% glycerol and 0.01% Ponceau S from the Ponceau S/glycerol stock solution
but no Coomassie blue dye to chromatographically pre-purified samples to avoid
dissociation of detergent-labile subunits.
Perform BN-PAGE at 4–7ºC, since broadening of bands is observed at room
temperature. Use cathode buffer B, and set power supply at 100 V until the sample
has entered the gel.
Continue electrophoresis with the current limited to 15 mA and voltage limited to
500 V (for gel dimension of 0.16 × 14 × 14 cm).
After the blue running front has moved about one-third of the desired total running
distance, remove cathode buffer B by suction pump (buffer B can be reused two or
three times). Continue the run using slightly blue cathode buffer B/10 for better
detection of faint protein bands, and to improve native blotting (less Coomassie dye
competes under these conditions with protein binding to PVDF membranes).
Stop electrophoresis after 2–4 h.
The BN gel can then be fixed and Coomassie and silver stained using any previously
published protocol for the staining of tricine-SDS gels. The migration distances of
individual bands relative to marker proteins can be used to estimate native masses of
membrane proteins.
References
Schagger H and von Jagow G (1991) Blue native electrophoresis for isolation of
membrane protein complexes in enzymatically active form. Anal Biochem 199: 223–231.
Schagger H, Cramer WA, von Jagow G (1994) Analysis of molecular masses and
oligomeric states of protein complexes by blue native electrophoresis and isolation of
membrane protein complexes by two-dimensional native electrophoresis. Anal Biochem
217: 220–230.
66
SDS-Polyacrylamide Gel Electrophoresis of Proteins
SDS-PAGE is the most widely used method for qualitatively analysing protein which is
based on the separation of proteins according to size. The velocity of charged molecule in
an electric field is inversely proportional to the frictional coefficient which in turn is
directly proportional to the molecular sizes. Electrophoretic mobility increases by 2% for
every 1oC rise in temperature. SDS is an anionic detergent and ~1.4g of SDS binds with
1g of protein. It gives uniform charges to mass ratio to the protein samples, so that the
movement of the protein on the gel will be totally based on the molecular masses.
Polyacrylamide Gels
Polymerization of acrylamide is an example of free-radical catalysis reaction and
is initiated by the addition of ammonium persulfate and the base TEMED which helps in
decomposition of persulphate ions to give free radicals.
Two types of gels are used in this method -
1. Separating gel (Resolving gel) (pH 8.8)
2. Stacking gel (pH 6.8)
Materials
1. Stock acrylamide solution: 30% acrylamide 0.8% bis-acrylamide. Filter through
Whatman No. 1 filter paper and store at 40C.
2. Buffers
Resolving gel buffer pH 8.8 (for 100 mL)
1.5 M Tris and 0.4% SDS (18.16 gm Tris, 0.4 gm SDS)
Stacking gel buffer pH 6.8 (for 100 mL)
0.5M Tris and 0.4% SDS (Wt. 6.05 gm Tris and 0.4 gm of SDS)
3. 10% ammonium persulfate. (Make fresh)
4. Electrophoresis buffer.
Dissolve 0.025 M Tris, 0.192 M glycine and 1% SDS. Make approx. 2 L of tank
buffer (pH 8.3)
5. Sample buffer (2X)
Stacking gel buffer : 2.5 mL
SDS 10% : 2.0 mL
Glycerol : 1.0 mL
β-mercaptoethanol : 0.25 mL
Bromophenol blue stock : 0.10 mL
6. Protein stain: 0.1% Commission brilliant blue R250 in 50% methanol, 10%
glacial acetic acid. Dissolve the dye in methanol and water component first, and
then add the acetic acid. Filter the final solution through Whatman No. 1 filter
paper if necessary.
7. Destain: 10% methanol, 7% glacial acetic acid in distilled water.
8. Microsyringe or micropipette tips.
67
Method
1. Samples to be run are first denatured in sample buffer by heating 95-100ºC for 5
min.
2. Mix the following:
Mix the following For 10% gel
Resolving gel buffer pH 8.8 8.0 mL
Water 8.0 mL
Stock-acrylamide 8.0 mL
Ammonium persulfate (10%) 120 μL
TEMED 35 μL
Total 24 mL
3. ‘Degas’ the solution under vacuum for about 30 second.

4. Using a Pasteur pipette transfer this separating gel mixture to the gel cassette until it
reaches a position 1 cm from the bottom of the comb that will form the loading
wells.
5. To ensure that the gel sets with a smoother surface very carefully put water or
butanol down one edge using a Pasteur pipette.
6. The gel can now be left to set.
7. While the separating gel is setting prepare the following stacking gel.
(For 5 mL)
Stacking gel buffer (pH 6.8) : 2.5 mL
Stock-acrylamide : 0.8 mL
Water : 1.6 mL
10% APS : 0.1 mL
TEMED : 5.0 μL
Degas the solution as before
8. When the separating gel has set, pour off the overlaying water/butanol. Add this
solution to the gel cassette and place the well forming comb into this solution and
leave to set making sure that no air bubbles are trapped.
9. Carefully remove the comb from the stacking gel after it sets and then rinse the
wells using buffer or clean the wells using small filter strips. Assemble the
cassette in the electrophoresis tank. Fill the top reservoir with electrophoresis
buffer and look for any leakage. If there is no leakage, fill the bottom tank with
electrophoresis buffer.
10. Load the samples (amount of protein to be loaded should be quantified) and equal
concentration of protein should be loaded in each well.
11. Connect the power pack to the apparatus and pass a current of 30 mA through the
gel (constant current). Ensure your electrodes have correct polarity. All proteins
will travel to the anode (+). Continue electrophoresis until the bromophenol blue
reaches the bottom of the gel.
12. Dismantle the gel apparatus, pry open the gel plates, remove the gel, discard the
stacking gel and place the separating gel in stain solutions.
68
13. Staining should be carried out with shaking. Stain is replaced with destain. After
destaining bands will appear.
References
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of
Bacteriophage T4. Nature 227:680–685.
69
Isoelectric Focusing for Separating the Protein Based
on Isoelectric Point (pI)
Isoelectric focusing is an electrophoretic method in which proteins are separated on the
basis of their pIs (1-12). It makes use of the property of proteins that their net charges are
determined by the pH of their local environments. Proteins carry positive, negative, or
zero net electrical charge, depending on the pH of their surroundings. The net charge of
any particular protein is the (signed) sum of all of its positive and negative charges. These
are determined by the ionisable acidic and basic side chains of the constituent amino
acids and prosthetic groups of the protein. If the number of acidic groups in a protein
exceeds the number of basic groups, the pI of that protein will be at a low pH value and
the protein is classified as being acidic. When the basic groups outnumber the acidic
groups in a protein, the pI will be high with the protein classified as basic. Proteins show
considerable variation in isoelectric points, but pI values usually fall in the range of pH 3-
12 with a great many having pI between pH 4.0 and pH 7.0.
Proteins are positively charged in solutions at pH values below their pI and

negatively charged above their isoelectric points. Thus, at pH values below the pI of a
particular protein, it will migrate toward the cathode during electrophoresis. At pH values
above its pI, a protein will move toward the anode. A protein at its isoelectric point will
not move in an electric field. When a protein is placed in a medium with a linear pH
gradient and subjected to an electric field, it will initially move toward the electrode with
the opposite charge (Figure 1). During migration through the pH gradient, the protein will
either pick up or lose protons. As it does, its net charge and mobility will decrease and
the protein will slow down. Eventually, the protein will arrive at the point in the pH
gradient equalling its pI. There, being uncharged, it will stop migrating. If a protein at its
pI should happen to diffuse to a region of lower pH, it will become protonated and be
forced toward the cathode by the electric field. If, on the other hand, it diffuses into a pH
higher than its pI, the protein will become negatively charged and will be driven toward
the anode. In this way, proteins condense, or focus, into sharp bands in the pH gradient at
their individual, characteristic pI values.
Figure 1. lsoelectric focusing. The motion of a protein undergoing isoelectric focusing is

depicted (circles). The protein is shown near its pI in a pH gradient. Both the pH
gradient and the motion of the protein are controlled by an electric field. At pH values
lower than the pI, the protein is positively charged (+) and it is driven toward the
cathode (arrow). Above the pI, the protein is negatively charged (-) and it moves toward
the anode. There is no net electrical force on the protein at its pI (0). The protein focuses
in a Gaussian distribution centered at the pI.
70
Focusing is a steady-state mechanism with regard to pH. Proteins approach their
respective pI values at differing rates but remain relatively fixed at those pH values for
extended periods. This type of motion is in contrast to conventional electrophoresis in
which proteins continue to move through the medium until the electric field is removed.
Moreover, in IEF, proteins migrate to their steady-state positions from anywhere in the
system. Thus, the sample application point is arbitrary. In fact, the sample can be initially
distributed throughout the entire separation system.
1. Establishing pH Gradients
Stable, linear pH gradients are the keys to successful IEF. Establishment of such
gradients is accomplished in two ways with two different types of molecules, carrier
ampholytes and acrylamido buffers. Carrier ampholytes (amphoteric electrolytes) are
mixtures of molecules containing multiple aliphatic amino and carboxylate groups. They
are small (about 300-1000 Da in size) multi-charged organic buffer molecules with
closely spaced pI values and high conductivity. Ampholytes are included directly in IEF
gels. In electric fields, carrier ampholytes partition into smooth pH gradients that increase
linearly from the anode to the cathode. The slope of a pH gradient is determined by the
pH interval covered by the carrier ampholyte mixture and the distance between the
electrodes. The use of carrier ampholytes is the most common and simplest means for
forming pH gradients.
Acrylamido buffers are derivatives of acrylamide containing both reactive double

bonds and buffering groups. Their general structure is CH2 = CH-CO-NH-R, where R
contains either a carboxyl [-COOH] or a tertiary amino group [e.g., -N(CH3)2]. They are
covalently incorporated into polyacrylamide gels at the time of casting. The key
acrylamido buffers have pK values at pH 1, 3.6, 4.6, 6.2, 7.0, 8.5, 9.3, 10.3, and >12.
They can be used to cast just about any conceivable pH gradients. In any given gradient,
some of the acrylamido compounds act as buffers while others serve as titrants. Because
the buffering compounds are fixed in place in the separation medium, the gels are called
"immobilized pH gradients", or IPGs. IPGs offer the advantage of gradient stability over
extended runs. They are, however, more cumbersome and expensive to cast than carrier
ampholyte gels. IPGs are commercially available in sheet form in a few pH ranges. A
greater variety of pH ranges are available in IPGs that have been cut into strips for the
IEF first dimension of 2-D PAGE.
IEF is a high-resolution technique that can routinely resolve proteins differing in

pI by less than 0.05 pH unit. Antibodies, antigens, and enzymes usually retain their
activities during IEF. The proper choice of ampholyte or IPG range is very important to
the success of a fractionation. Ideally, the pH range covered by an IEF gel should be
centered on the pI of the proteins of interest. This ensures that the proteins of interest
focus in the linear part of the gradient with many extraneous proteins excluded from the
separation zone.
With carrier ampholytes, concentrations of about 2% (w/v) are best. Ampholyte

concentrations below 1% (w/v) often result in unstable pH gradients. At concentrations
above 3% (w/v) ampholytes are difficult to remove from gels and can interfere with
protein staining. When casting IPGs, follow published recipes and use buffering powers
of about 3 meq throughout the gradient.
71
2. Gels for Isoelectric Focusing
As an analytical tool, IEF is carried out in large-pore polyacrylamide gels (5%T,

3%C) which serve mainly as anti-convective matrices. Polyacrylamide IEF gels are
polymerized with an initiator system including riboflavin for photopolymerization.
Photochemical initiation of polymerization with a combination of the three compounds
riboflavin, ammonium persulfate, and TEMED, results in more complete polymerization
of IEF gels than chemical polymerization does in gels containing low-pH ampholytes.
Suitable initiator concentrations are 0.015% ammonium persulfate, 0.05% TEMED, and
5 µg/mL riboflavin-5'-phosphate. Photochemical polymerization is allowed to continue
for 2 hr, with the second hour under direct lighting from a nearby fluorescent lamp.
The most common configuration for analytical IEF is the horizontal

polyacrylamide slab gel. Gels are cast with one exposed face on glass plates or specially
treated plastic sheets. They are placed on cooling platforms and run with the exposed face
upward. Electrolyte strips, saturated with 0.1-1 M phosphoric acid at the anode and 0.1-1
M sodium hydroxide at the cathode, are placed directly on the exposed surface of the IEF
gel. Electrodes of platinum wire maintain contact between the electrical power supply
and the electrolyte strips. In another possible configuration, the gel and its backing plate
are inverted and suspended between two carbon rod electrodes without the use of
electrolyte strips. IPG strips for 2-D PAGE are often run with the gel facing down in
dedicated IEF cells.
Protocol 1. Casting gels for isoelectric focusing

Equipment and reagents
Gel casting apparatus for a horizontal electrophoresis cell; e.g., the GE MultiPhor
Fluorescent lamp
30%T, 3%C acrylamide stock solution
50% glycerol
0.1% riboflavin-5'-phosphate (FMN)
Carrier ampholytes with a pH range spanning the pIs of the proteins of interest
10% APS and TEMED
Procedure
The formulation given here is for 12 mL of gel solution containing 5% T (3%C)
acrylamide, 2% carrier ampholytes, 5% glycerol. The volume needed depends on the
casting apparatus that is used; adjust volumes accordingly. This recipe is sufficient for
casting one gel of 100 x 125 x 0.8 mm (10 mL) or four 100 x 125 x 0.2 mm (10 mL
total). (8 M urea can be substituted for the glycerol if desired)
1. Assemble the casting apparatus according to the manufacturer's instructions. The

use of gel support film for polyacrylamide is highly recommended.
2. Combine -
30%T, 3%C acrylamide stock Carrier 2.0 mL
Carrier ampholytes (40%) 0.6 mL
50% glycerol 1.2 mL
Water 8.2 mL
0.1% FMN
72
10% APS
TEMED
Swirl the solution gently to mix the components.
3. Transfer the gel solution to the casting apparatus with a pipette and bulb.
4. Position a fluorescent lamp about 3-4 cm from the gel solution and illuminate the
solution for about one hour.
5. Open the gel cassette or lift the gel from the casting tray to expose the face of the
gel. Place the gel with the open face upward and illuminate it with the fluorescent
lamp for an additional 30 min.
6. The gel may be used immediately or it can be covered with plastic wrap and
stored at 4°C for several days. Best results are sometimes obtained when IEF gels
are left overnight at 4°C before use.
To prepare 30%T, 3% C acrylamide solution, dissolve 29.1 g of acrylamide and
0.9 g of bisacrylamide in 72.5 mL of water. The final volume will be 100 mL.
Ultrathin gels (<0.5 mm) allow the highest field strengths and, therefore, the
highest resolution of the analytical methods. Electro focusing can also be done in tubes,
and this configuration once constituted the first dimension of 2-D PAGE. Because of
difficulties in handling and reproducibility with tube gels, IPG strips have largely
replaced them. Good visualization of individual bands generally requires a minimum of
0.5 µg each with dye staining or 50 ng each with silver staining. One of the simplest
methods for applying samples to thin polyacrylamide gels is to place filter paper strips
impregnated with sample directly on the gel surface. A convenient size for applicator
papers is 0.2 x 1 cm, holding 5 ng of sample solution. Alternatively, 1- to 2- µL samples
can be placed directly on the surface of the gel. In most cases, IPG strips (which are
provided in dehydrated form) are rehydrated in sample-containing solution prior to
electrophoresis. Rehydration loading allows higher protein loads to be applied to gels
than do other methods. It is particularly popular because of its simplicity.
There are no fixed rules regarding the positioning of the sample on the gel. In
general, samples should not be applied to areas where they are expected to focus. To
protect the proteins from exposure to extreme pH, the samples should not be applied
closer than 1 cm from either electrode. Forming the pH gradient before sample
application also limits the exposure of proteins to pH extremes. Precast IEF mini gels (6
cm long by 8 cm wide and 1 mm thick) are available for carrying out carrier-ampholyte
electro focusing. A selection of IPG sheets is also available for horizontal IEF. Vertical
IEF gels have the advantage that the electrophoresis equipment for running them is
available in most laboratories and they can hold relatively large sample volumes. Because
vertical electrophoresis cells cannot tolerate very high voltages, this orientation is not
capable of the ultrahigh resolution of horizontal cells. To protect the materials of the
electrophoresis cells (mainly the gaskets) from caustic electrolytes alternative catholyte
and anolyte solutions are substituted in vertical IEF runs. As catholyte, 20 mM arginine,
20 mM lysine is recommended in vertical slab systems (0.34 g arginine free base and
0.36 lysine free base in 100 mL of water). The recommended anolyte is 70 mM H3PO4,
but it can be substituted with 20 mM aspartic acid, 20 mM glutamic acid (0.26 g aspartic
acid and 0.29 g glutamic acid in 100 mL of water).
73
3. Power conditions and resolution in isoelectric focusing
The pH gradient and the applied electric field determine the resolution of an IEF run.
According to theory and experiment, the difference in pI between two resolved adjacent
protein IEF bands is directly proportional to the square root of the pH gradient and
inversely proportional to the square root of the voltage gradient (field strength) at the
position of the bands. In addition to the effect on resolution, high electric fields also
result in shortened run times. However, high voltages in electrophoresis are accompanied
by large amounts of generated heat. Thus, there are limitations on the magnitudes of the
electric fields that can be applied and the ionic strengths of the solutions used in IEF.
Because of their higher surface-to-volume ratio, thin gels are better able to dissipate heat
than thick ones and are therefore capable of higher resolution (high voltage). Electric
fields used in IEF are generally of the order of 100 V/cm. At focusing, currents drop to
nearly zero since the current carriers have stopped moving by then.
4. Protein Solubilisation for Isoelectric Focusing
A fundamental problem with IEF is that some proteins tend to precipitate at their pI
values. Carrier ampholytes sometimes help overcome pI precipitation and they are
usually included in the sample solutions for IPG strips. In addition, non-ionic detergents
or urea are often included in IEF runs to minimize protein precipitation. Urea is a
common solubilizing agent in gel electrophoresis. It is particularly useful in IEF,
especially for those proteins that tend to aggregate at their pIs. Urea disrupts hydrogen
bonds and is used in situations in which hydrogen bonding can cause unwanted
aggregation or formation of secondary structures that affect the mobility. Dissociation of
hydrogen bonds requires high urea concentrations (7-8 M). If complete denaturation of
proteins is sought, samples must be treated with a thiol-reducing agent to break disulfide
bridges (protein solutions in urea should not be heated above 30°C to avoid
carbamylation).
High concentrations of urea make gels behave as if they had reduced pore sizes.
This is because of either viscosity effects or reductions in the effective size of water
channels (pores). Urea must be present in the gels during electrophoresis, but, unlike
SDS, urea does not affect the intrinsic charge of the sample polypeptides. Urea solutions
should be used soon after they are made or treated with a mixed-bed ion-exchange resin
to avoid protein carbamylation by cyanate in old urea. Some proteins, especially
membrane proteins, require detergent solubilisation during isolation. Ionic detergents,
such as SDS, are not compatible with IEF, although non-ionic detergents, such as
octylglucoside, and zwitterion detergents, such as 24(3- cholamidopropyl)
dimethylammonio]-1-propane-sulfonate (CHAPS) and its hydroxyl analog CHAPSO, can
be used. NP-40 and Triton X-100 sometimes perform satisfactorily, but some
preparations may contain charged contaminants. Concentrations of CHAPS and
CHAPSO or of octylglucoside of 1-2% in the gel are recommended. Some proteins may
require as high as 4% detergent for solubility. Even in the presence of detergents, some
samples may have stringent salt requirements. Salt should be present in a sample only if it
is an absolute requirement. Carrier ampholytes contribute to the ionic strength of the
solution and can help to counteract a lack of salts in a sample. Small samples (1 to 10 pI)
in typical biochemical buffers are usually tolerated, but better results can be obtained
74
with solutions in deionized water, 2% ampholytes, or 1% glycine. Suitable samples can
be prepared by dialysis or gel filtration.
Protocol 2. Isoelectric focusing

Equipment and reagents
Flat-bed electrophoresis cell; e.g., the GE Multiphor
Power supply capable of delivering 2-3000 V and 6 Watt high voltage.
Refrigerated water circulator if required
1 N NaOH catholyte if required
1 N H3PO4 anolyte if required
Electrode strips if required
Sample application strips
Procedure
1. Set up the IEF cell as recommended by the manufacturer. This includes
connecting a water circulator, if used, and preparing the cooling platform and
electrode strips, if necessary.
2. Place sample application strips on a glass plate and pipette 5 µL of a protein
sample to each strip. Place the application strips 1 cm from the anode end of the
gel.
3. Position the gel in the IEF cell and make electrode contact as specified for the
particular cell.
4. Close the electrophoresis cell and connect the leads to the power supply; the red
lead is the anode and the black lead is the cathode.
5. Set the running conditions as recommended by the manufacturer of the
electrophoresis cell.
To prepare 1 N NaOH, dissolve 4 g of NaOH in 100 mL of water. To prepare 1 N
H3PO4, dissolve 2.3 mL of 85% H3PO4 (14.6 M, 44 N) in 97.7 mL of water. For
electrode strips, cut thick filter paper about 7 mm wide and about 4 mm shorter
than the gel. For sample application strips, cut thin filter paper to 0.2 x 1 cm, one
strip per sample.
References
Andrews AT (1986) Electrophoresis: theory, techniques, and biochemical and clinical
applications (2nd edn). Oxford University Press, Oxford.
Laas T (1989) In Protein purJI cation: principles, high resolution methods and
applications (ed. J.-C. Janson and L. Ryden), p.376. VCH Press, Weinheim. Dunn MJ
(1993) Gel electrophoresis: proteins. BIOS Scientific Publishers, Oxford.
75
Two-Dimensional Gel-Electrophoresis of Proteins
Two-dimensional gel electrophoresis was first introduced by P. H. O’Farrell and J. Klose
in the year 1975. Two-dimensional electrophoresis (2-D electrophoresis) is a powerful
and widely used method for the analysis of complex protein mixtures extracted from
cells, tissues, or other biological samples. These technique sorts proteins according to two
independent properties in two discrete steps: the first dimension step, isoelectric focusing
(IEF), separates proteins according to their isoelectric points (pI); the second dimension
step SDS-polyacrylamide gel electrophoresis (SDS-PAGE), separates proteins according
to their relative molecular weights. Each spot on the resulting two-dimensional array
corresponds to a single protein species in the sample. Thousands of different proteins can
thus be separated, and information such as the protein pI, the apparent molecular weight,
and the amount of each protein is obtained.
2-D Gel Electrophoresis Workflow
1. Sample preparation
2. Estimation of protein concentration
3. Protein Purification
4. 1-D Isoelectric Focusing
5. 2-D SDS-PAGE
6. Staining
7. Destaining
Procedure
1. Sample preparation using 2Dxtract kit of G-Biosciences
Grind 1g sample to the fine powder using liquid nitrogen.
Prepare 2 mL 2Dxtract by mixing 1 g dry 2D xtract and 1.1 mL diluent III.
Add this to the fine powder of sample.
Vortex and incubate at room temperature for 10- 15 minutes.
Centrifuge at 13000 rpm for 30 min at 4°C.
Collect supernatant in a fresh tube.
2. Protein estimation by Bradford’s method

Prepare BSA standards, blank and sample.
Blank: Add 1 mL double distilled water to the 2 mL Bradford reagent.
Sample: Add 10 µL Protein extract to 990 µL of double distilled water and 2
mL Bradford’s reagent.
Keep them at room temperature for 10 min.
Take OD at 595 nm wavelength.
Calculate the protein concentration from standard graph.
3. Protein purification using Bio-Rad 2-D cleanup kit

Transfer upto 500 µg of protein in a final volume of 100 µL into a 1.5 mL
micro centrifuge tube.
Add 300 µL precipitating agent 1 to the protein sample and mix well by
vortexing. Incubate on ice for 15 min.
Note: When adding solution, do not touch protein sample with the pipette tip. The protein may
precipitate on the tip causing sample loss.
76
Add 300 µL precipitating agent 2 to the mixture of protein and precipitating
agent 1. Mix well by vortexing.
Centrifuge the tube(s) at maximum speed (>12,000 rpm) for 5 min to form a
tight pellet.
Without disturbing the pellet, remove and discard the supernatant using a
pipette.
Position the tube in the centrifuge as before and centrifuge for 15–30 sec to
collect any residual liquid at the bottom of the tube. Use a pipette to carefully
remove the remaining supernatant.
Add 40 µL of wash reagent 1 on top of the pellet. Position the tube in the
centrifuge as before and centrifuge at maximum speed (>12,000 rpm) for 5
min.
Note: A precipitate may form along the tube wall. In these cases, vortex and/or pipette wash
solution over the pellet several times to ensure entire pellet is thoroughly washed.
Remove and discard the wash.
Add 25 µL of ReadyPrep proteomic grade water. Vortex the tube for 10–20s
Protein pellets may disperse, but will not dissolve in the water.
Add 1 mL of wash reagent 2 (pre-chilled at -20°C for at least 1 hr) and 5 µL of
wash 2 additive. Vortex the tube for 1 min.
Incubate the tube at -20°C for 30 min. Vortex the tube for 30 sec every 10 min
during the incubation period.
After the incubation period, centrifuge the tube at top speed for 5 min to form a
tight pellet. Remove and discard the supernatant. Centrifuge the tube briefly
(15–30 sec). The pellet will appear white at this stage.
Air-dry the pellet at room temperature for no more than 5 min.
Note: Do not over-dry pellets. Over-dried pellets will be difficult to re-suspend.
Re-suspend each pellet by adding an appropriate volume of 2-D
rehydration/sample buffer to the pellet.
4. First dimension Separation: Isoelectric Focusing (IEF)

Take appropriate volume of protein sample dissolved in rehydration buffer which
is used to denature and solubilize protein samples, and rehydrate the IPG strips as
indicated below –
IPG Strip length (cm) Total volume per strip (µL)

7 125
11 200
13 250
18 340
24 450
Add 10 µL of bromophenol blue to the sample. Place the sample in the centre of
rehydration tray.
Remove the protective cover from the IPG strip starting at the positive end.
Position the IPG strip over the sample with the gel side down in such a way that
there is no air bubble.
Apply mineral oil over the strip to minimize the evaporation.
Wrap the tray with aluminium foil and leave it overnight at room temperature.
Next morning, clean the focusing tray and transfer the strip from rehydration tray
to focusing tray. This time, gel side of the strip should face upwards.
77
Take 2 wicks, dip them in distilled water and place them at the ends of the strip.
Set both the electrodes and programme according to the size of strip.
Run the programme.
Once the program is complete, turn off the power and remove the strips.
For immediate use, go for washing step otherwise strips can be stored at -80°c.
Washing
Dip the IPG strip in equilibrium buffer I for 10 min on rocker. Discard buffer I
and replace it with equilibrium buffer II. Keep it for 10 min on rocker.
Discard buffer II and wash the strip with 1 X SDS PAGE running buffer.
5. Second dimension Separation – (SDS-PAGE)
Make a 10 % SDS polyacrylamide gel without the upper stack, leaving about 50 mm
of room from the top.
Place the IPG strip over the SDS-PAGE gel in such a way that gel side of the strip
faces front side.
Seal the strip with 1% agarose prepared in 1 SDS running buffer.
Load a protein marker at one end of the gel. Run the unit at 18-20 mA.
6. Staining
One can go either for silver staining or coomassie brilliant blue (CBB) staining.
CBB Reagent - 0.1% dye
10% acetic acid
50% methanol and make up the volume to 100 mL
Dip the PAGE gel in staining solution for approx. 1 h. After staining remove the
staining solution.
7. Destaining: For preparation of 100 mL destaining solution -
Add 7 mL glacial acetic acid to 30 mL of methanol and make up the volume to

100 mL.
Dip the stained gel in destaining solution and keep it on rocker for 10-15 minutes.
Repeat this procedure until the spots become visible (The background should
become clear).
Compare the results by performing image analysis.
78
Preparation of solutions -
A. Rehydration Buffer: 10 mL
Solution Component Amount
Urea 4.2 g
Thiourea 1.52 g
CHAPS 0.4 g
1 M DTT 500 µL
100% IPG buffer (Ampholines) 50 µL
1% Bromophenol blue 200 µL
Deionized water (Milli Q) Up to 10 mL
Note: Do not add DTT or IPG buffer until ready to use.
B. SDS Equilibration Buffer – (100 mL)

1 M Tris, pH 8.8 5 mL
Urea 36.03 g
100% Glycerol 30 mL
10% SDS 20 mL
1% Bromophenol Blue 200 µL
Deionized water (Milli Q) to 100 mL
For equilibration buffer I, add 100 mg DTT to 10 mL of stock SDS
equilibration buffer. For equilibration buffer II, add 250 mg IAA
(Iodoacetamide) to 10 mL of stock SDS equilibration buffer.
C. 10% SDS Polyacrylamide Gel -

Deionized water (Milli Q) 2.0 mL
30% Acrylamide 2.0 mL
Resolving buffer pH 8.8(1X) 2.0 mL
10% APS 32 µL
TEMED 10 µL
References
Dunn MJ (1993) Gel electrophoresis: proteins. BIOS Scientific Publishers, Oxford.

Garfin DE (1995) In Introduction to biophysical methods for protein and nucleic acid
research (ed. J.A. Glasel and M.P. Deutscher), p. 53. Academic Press, San Diego.
79
Antioxidant Enzymes and their Activity Assay
Suneha Goswami, Ranjeet R. Kumar and Shelly Praveen

110012, India
Introduction
In a biological context, reactive oxygen species (ROS) are produced as a natural
byproduct of the normal metabolism of oxygen and have important roles in many aerobic
cellular metabolic processes such as cell signalling and homeostasis. However, during
times of abiotic/biotic stress (e.g., UV, heat exposure, drought, pathogen infection), ROS
levels can increase dramatically. Increased levels of ROS are cytotoxic, while lower
levels are necessary for the regulation of several key physiological mechanisms including
apoptosis, cell differentiation, cell proliferation and regulation of redox-sensitive signal
transduction pathways. However, increased levels can also result in ROS-induced
damage including mutations, chromosomal aberrations, carcinogenesis and cell death.
Cells contain a large number of antioxidant enzymes to prevent or repair the damage
caused by ROS, as well as to regulate redox-sensitive signalling pathways.
Antioxidant enzymes
The intracellular concentration of ROS depends on the production and/or removal by the
antioxidant system. Cells contain a large number of antioxidants to prevent or repair the
damage caused by ROS, as well as to regulate redox-sensitive signalling pathways. Three
of the primary antioxidant enzymes contained in living cells that are thought to be
necessary for life in all oxygen metabolizing cells are superoxide dismutase (SOD),
catalase, and a substrate specific peroxidase. The SODs convert superoxide radical into
hydrogen peroxide (H2O2) and molecular oxygen (O2), while the catalase and peroxidases
convert hydrogen peroxide into water and in the case of catalase to oxygen and water.
The net result is that two potentially harmful species, superoxide radicals and hydrogen
peroxide, are converted to water. There are three SOD enzymes that are highly
compartmentalized. Manganese-containing superoxide dismutase (MnSOD) is localized
in the mitochondria; copper- and zinc-containing superoxide dismutase (CuZnSOD) is
located in the cytoplasm and nucleus and extracellular SOD is expressed extracellularly
in some tissues. Other compartmentalized antioxidant enzymes include catalase, which is
found in peroxisomes and cytoplasm, and GPx, which can be found in many sub-cellular
compartments including the mitochondria and nucleus depending on the family member.
Thus, the many forms of each of these enzymes reduce oxidative stress in the various
parts of the cell. Thus, antioxidant proteins with similar enzymatic activity may have
different effects after modulation due to different localizations within cells.
Assays for measuring antioxidant enzymes

There are two different methods to determine the activity of antioxidant enzymes – (a)
Enzyme activity assay, and (b) In-gel activity assay. The enzyme activity assay
determines the biological impact of the antioxidant enzymes. Since, the expression of the
antioxidant enzyme mRNA or protein does not necessarily result in an increase in
activity, enzymatic assays and native gels are utilized to measure the activity of the
80
antioxidant enzymes. The activity assay requires 10-fold more protein than the in-gel
assays but gives a quantitative result while the native gel requires less protein but results
in a qualitative result. Additionally a visual image is often a compelling way to present or
address a scientific question.
81
Superoxide Dismutase enzyme activity assay
Superoxide dismutases (SOD; EC 1.15.1.1) are metalloproteins catalysing the
dismutation of superoxide free radical to molecular oxygen and H2O2. The reaction may
be written as follows:
O2•− + O2 •− + 2H+ H2O2 + O2
Thus superoxide dismutase provides defence against the oxygen toxicity caused by the
superoxide radicals. The metalloprotein with this enzyme activity may have Cu-Zn or Mn
or Fe as its cofactor.
Principle
Because of the instability of its substrate, all available assays of superoxide dismutase are
indirect and depend upon its ability to scavenge O2•− from reaction mixture and thus to
inhibit the reaction caused by O2•− . In the biochemical method, xanthine-xanthine
oxidase is used to generate O2 •− and nitroblue tetrazolium (NBT) reduction is used as an
indicator of O2 •− production. SOD will compete with NBT for O2 •−; the percent
inhibition of NBT reduction is a measure of the amount of SOD present.
Reagents
(i) Methionine solution (200 mM)

Dissolve 300 mg of methionine (mol. wt. 150) in 100 ml of double distilled water.
(ii) Riboflavin solution (150 mM)
Dissolve 28.2 mg riboflavin (mol. wt. 376) initially in 2 ml of 0.1 N NaOH and
then make up the volume to 100 ml. Take 10 ml out of this and make it to 50 ml.
(iii)Nitro-blue tetrazolium solution (2.250 mM)
Dissolve 92 mg of NBT (mol. wt. 817) in 50 ml of double distilled water.
(iv) EDTA disodium solution (1.5 mM)
Dissolve 56 mg of EDTA-disodium salt (mol. wt. 372) in 100 ml of double
distilled H2O.
(v) Phosphate Buffer (50 mM; pH 7.0)
Dissolve 0.265 g of KH2PO4 (mol. wt. 136) and 0.53 g of K2HPO4 (mol. wt. 174)
in 50 ml of distilled water. Adjust the pH to 7.0, if there is need to do so, and
make up the volume to 100 ml with distilled water.
(vi) Phosphate Buffer (50 mM; pH 7.8)
Dissolve 0.058 g of KH2PO4 (mol. wt. 136) and 0.8 of K2HPO4 (mol. wt. 174) in
50 ml of distilled water. Adjust the pH to 7.8, if there is need to do so, and make
up the volume to 100 ml with distilled water.
Procedure
Preparation of Enzyme extract
Plant material is cut into small pieces and homogenized in cold 50 mM Phosphate
buffer (pH 7.0).
The homogenate is centrifuged at 13,000 rpm for 30 min and the supernatant
obtained is used as enzyme extract.
82
Enzyme assay
Prepare a reaction mixture (RM) as follows

S. No. Sample Methionine NBT EDTA Enzyme PO4 Buffer Riboflavin
(200 mM) (2.250 (1.5 mM) extract (50 mM) (75 µM)
(ml) mM) (ml) (pH 7.8) (ml)
(ml) (ml)
1. Control 0.2 0.2 0.2 - 2.200 0.2
2. S1 0.2 0.2 0.2 10 2.190 0.2
3. S2 0.2 0.2 0.2 20 2.180 0.2
4. S3 0.2 0.2 0.2 30 2.170 0.2
5. S4 0.2 0.2 0.2 40 2.160 0.2
Riboflavin is added as the last component and the tubes are shaken and placed 30
cm below a light bank consisting of two 15-W fluorescent tubes.
The reaction is started by switching on the light and is allowed to run for 10 min.
After 10 min the reaction is stopped by switching off the light.
The tubes are covered with black cloth immediately after switching off the light.
Absorbance of the reaction mixture is read at 560 nM.
Note : A non-irradiated reaction mixture has zero absorbance at 560 nm, while the reaction
mixture lacking enzyme develops the most colour. The colour decreases with increasing volume of
extract added.
Log A560 is plotted as a function of the volume of the enzyme extract in the
reaction mixture.
The volume of the enzyme extract producing 50% inhibition of the reaction is
read from the resultant graph.
One unit of the SOD activity is defined as the enzyme which causes 50%
inhibition of NBT reduction in one minute and specific activity of SOD is
reported as units per mg protein.
83
In-gel activity assay of SOD
1. Clean glass plates with methanol or ethanol to rid the surface of any debris, making
sure there are no cracks or chips in the plates. Assemble the plates in the gel clamp
apparatus and stand using 1.5 mm spacers, thus making a “thick” gel.
2. For SOD stained gels, run duplicate samples on two 12% (v/v) native gels. Add all
reagents together in a 50 ml conical tube and mix well. Add the solution to the glass
plate assembly about 1 cm from the top of the apparatus using a 5 ¾” glass Pasteur
pipette. Slowly add a layer of water over the running gel until it is entirely covered.
Make sure the apparatus is level and wait 20 to 30 min for polymerization of the gel.
An interface can clearly be seen between the water layer and the gel when
polymerization is complete.
3. Stacking gel preparation: After the separating gel has polymerized, prepare a 5%
(v/v) stacking. Mix all reagents together in a 50 ml conical tube. Add to the surface of
the running gel, filling the glass assembly to the top with the stacking gel. Add a 1.5
mm comb, at an angle to avoid air bubbles under the comb surface; this is critical for
the proper running of your samples. Place the gel apparatus under a fluorescent light
or on a light box to aid in the polymerization reaction. Under a light, polymerization
will occur within 15-30 min.
4. Pre-Electrophoresis. Once the gel is polymerized, remove the combs and the gel
assemblies from the casting stand. Attach gel assemblies to the electrode assembly
and place into the electrophoresis apparatus. Pour pre-electrophoresis buffer into the
reservoir and chamber of the apparatus and add the apparatus cell lid. Pre-
electrophorese the gels for 1 h, at 40 mA at 4 °C. For best results, leave the gel
overnight at 4 °C.
Critical STEP The pre-electrophoresis step removes residual APS, TEMED, and incomplete
polymerization products which may inactivate native proteins.
5. Loading the gel. Leave the gels in the pre-electrophoresis buffer. Clean the wells with
a 5 ¾” glass Pasteur pipette to remove residual gel reagents. Prepare the samples, by
diluting the stock sample 1:1 in loading gel buffer and add the samples to the wells.
For samples prepared, load 50 – 250 μg protein per sample. Typically 150 μg will
give distinct bands. For tissue homogenates, load 50 – 100 μg protein per sample.
6. Electrophoresis. Run the samples in the pre-electrophoresis buffer for 3 h at 40 mA at
4 °C. After 3 h, remove the gel set-up and pour off the pre-electrophoresis buffer.
PAUSE POINT: The gels can be left at 4 °C for 18-24 h.
7. Add the electrophoresis buffer to the reservoir and chamber of the apparatus. Run the
gel for about 2-3 h more (40 mA, 4 °C). To ensure the proteins have entered the gel,
watch for a dye line at the top of the sample dye front. Once the dye front reaches the
bottom of the gel, run the gel for 1 h more.
8. Stain gels with SOD native gel stain. Place one gel in a plastic or glass container
containing 40 ml of total SOD stain and the other gel in 40 ml of MnSOD stain. Stain
for 20 min at room temperature, shaking in the dark or covered by foil.
9. Following incubation, rinse the gels gently with ddH2O twice. Add enough ddH2O to
cover the gel and place under a fluorescent light and on a light box. The gel will begin
to turn blue/purple and clear bands should appear gradually. Leave under light, and
on the light box for 15 min to 2 h making sure the gel does not dry out.
84
10. After bands have appeared, wash three times for 10 min with water. Allow the gel to
sit at room temperature for 18-24 h in water under ambient light to allow for further
band development.
11. Turn off the light. The bands will intensify over the next 4 to 24 h. Achromatic bands
indicate the presence of SOD.
12. Image the gels on a computer flat-bed scanner with light from top and bottom or
utilize other available imaging systems.
References
Beanchamp, C. and Fridovich, I. (1971) Superoxide dismutase : Improved assays and

assay applicable to acrylamide gels. Anal. Biochem. 44 : 276-287.
Fridovich, I. (1975). Superoxide dimutase. Ann. Rev. Biochem. 44 : 147-159. Christine J.
Weydert and Joseph J. Cullen (2010) Nat Protoc. 5(1): 51–66.
doi:10.1038/nprot.2009.197.
85
Catalase enzyme activity assay
Catalase activity was assayed following the method of Luck (1974). Catalase converts
hydrogen peroxide to water and oxygen. Catalase activity is largely located in subcellular
organelles known as peroxisomes, mitochondria but it is absent in the chloroplast.
H2O2 H2 O + O2
Principle
The UV absorption of hydrogen peroxide can be measured at 240nm, whose absorbance
decreases when degraded by the enzyme catalase. From the decrease in absorbance, the
enzyme activity can be calculated.
Reagents
1. Phosphate buffer: 0.067 M (pH 7.0)
2. Hydrogen peroxide (2mM) in phosphate buffer
Procedure
Preparation of enzyme extract
Same as for SOD enzyme extract preparation.
Enzyme assay

H2O2-phosphate buffer (3.0 mL) taken in an experimental cuvette.

Add 40 µL of enzyme extract and mixed thoroughly.

The time required for a decrease in absorbance by 0.05 units will be recorded at 240
nm in a spectrophotometer.

The enzyme solution containing H2O2-free phosphate buffer served as control.

One enzyme unit will be calculated as the amount of enzyme required to decrease
the absorbance at 240 nm by 0.05 units.
Enzyme activity calculation

The concentration of H2O2 can be calculated from absorbance using the following
expression:
[H2O2 mM] = (Absorbance 240nm × 1000) / 39.4mmol-1 cm-1
Where 39.4 mmol-1 cm-1 is the molar extinction coefficient for H2O2.
86
In-gel activity assay of Catalase
1. For catalase gels prepare 8% (v/v) native gel. Only one gel is needed for each
antioxidant enzyme.
Critical STEP Run gels at 4 °C. All staining steps are performed at room temperature.
2. Prepare the samples by diluting the stock 1:1 in loading gel buffer and add the
samples to the wells. For tissue homogenates, load 50-100 µg protein per sample.
Load a positive control for catalase (bovine catalase, 10 mU) or GPx (bovine or
human GPx, 50 mU).
3. Prepare and run all other aspects of the assay as for the SOD activity gel (see
Steps 1-7).
4. Remove the gel from the glass plates and place into a glass staining dish.
5. Wash the gel 3 × 10 min in distilled water.
6. Prepare a 0.003% H2O2 solution by mixing 10 µL of H2O2 (30% solution, v/v)
with 100 mL of ddH2O in a glass beaker with a stir bar. Incubate the gel in the
0.003% H2O2 (v/v) for 10 min.
7. Prepare the stain in two 50 ml conical tubes; by making a 2% ferric chloride (w/v,
0.6 gm in 30 mL distilled water) in one tube and in a second tube preparing 2%
potassium ferricyanide (w/v, 0.6 gm in 30 ml distilled water).
8. Rinse the gel from Step 6 gel twice with ddH2O twice for 5 min. Pour off water.
9. Add the stain to gel.
CAUTION Ferric chloride and potassium ferricyanide are caustic and will leave an indelible stain
– USE GLOVES!
CRITICAL STEP DO NOT MIX the 2 reagents prior to staining, pour them together directly on
top of the gel.
10. When achromatic bands begin to form, pour off the stain and rinse extensively
with ddH2O.
11. Image as described in SOD activity gel (Steps 9-12).
87
Guaiacol Peroxidase (POD)
Guaiacol peroxidase (Guaiacol: H2O2 oxidoreductase, EC 1.11.1.7) activity can be measured
according to Gasper et al. (1975).
Guaiacol + H2O2 Tetraguaiacol + H2O
Principle
The absorption of tetraguaiacol can be measured at 420nm, whose absorbance increase when
guaiacol reacts with H2O2 in the presence of the enzyme guaiacol peroxidase. From the
increase in absorbance, the enzyme activity can be calculated.
Reagents
1. 50 mM potassium phosphate buffer (pH 6.1),
2. 1% guaiacol
3. 0.4% H2O2
4. Enzyme extract
Procedure
Preparation of enzyme extract
Same as for SOD enzyme extract preparation.
Enzyme assay
 Take phosphate buffer (3.0ml) in an experimental test tube.
 Add 1% guaiacol, 40µl of enzyme extract and 0.4% H2O2, and mixed thoroughly.
 Increase in the absorbance due to oxidation of guaiacol will be measured at 470 nm.
 The enzyme solution containing guaiacol-free phosphate buffer served as control.
 One enzyme unit will be calculated as the amount of enzyme required to increase in
the absorbance at 470nm by 0.05 units.
 Enzyme activity will be measured in terms of mmol of guaiacol oxidized min-1 g-1
fresh weight.
Calculation of enzyme activity

The concentration of tetraguaiacol can be calculated from the increase in absorbance using
the following expression:
[Tetraguaiacol mM] = (Absorbance 470nm × 1000) / 26.6 mmol-1 cm-1
where 26.6 nmol-1 cm-1 is the molar extinction coefficient for tetraguaiacol.
88
In-gel Activity Assay of Guaiacol Peroxidase
1. Prepare the gel and run the enzyme extract as mentioned in case of in-gel activity
assay of catalase.
2. After complete run, wash the gel with water three times, 10 min each washing.
3. Immersed the gel in 0.018M guaiacol for 30 min at room temperature.
4. Rinsed the gel twice with ddH2O and immersed in 0.015% H2O2 in 1% glacial acetic
acid till the development of dark brown bands.
89
Agarose Gel Electrophoresis
Vinutha T.
110012, India.
Introduction
Agarose gel electrophoresis is the easiest and most popular way of separating and analyzing
DNA. Here DNA molecules are separated on the basis of charge by applying an electric field
to the electrophoretic apparatus. Shorter molecules migrate more easily and move faster than
longer molecules through the pores of the gel and this process is called sieving. The gel
might be used to the DNA in order to quantify it or to isolate a particular band. The DNA can
be visualized in the gel by the addition of ethidium bromide.
Agarose is a polysaccharide obtained from the red algae Porphyra umbilicalis. Its
systematic name is (1 4)-3, 6-anhydro-a-L-galactopyranosyl-(1 3)-β-D-galactopyranan.
Agarose makes an inert matrix. Most agarose gels are made between 0.7% and 2% of
agarose. A 0.7% gel will show good separation for large DNA fragments (5-10kb) and a 2%
gel will show good resolution for small fragments with size range of 0.2-1kb. Low percentage
gels are very weak (Note:- it may break when you lift them) but high percentage gels are
usually brittle and do not set evenly. The volume of agarose required for a minigel
preparation is around 30-50ml and for a larger gel, it is around 250ml.
Figure: Structure of agarose

Factors affecting the movement of DNA
1. Voltage applied
The migration rate of the linear DNA fragments through agarose gel is proportional to
the voltage applied to the system. As voltage increases, the speed of DNA also
increases. But voltage should be limited because it heats and finally causes the gel to
melt.
2. Ethidium bromide (EtBr)
It is an intercalating agent which intercalates between nucleic acid bases and allows
the convenient detection of DNA fragments in gel. When exposed to UV light, it will
fluoresce with an orange colour. After the running of DNA through an EtBr-treated
90
gel, any band containing more than ~20 ng DNA becomes distinctly visible under UV
light. EtBr is a known "mutagen", however, safer alternatives are available. It can be
incorporated with agarose gels or DNA samples before loading, for visualization of
the fragments. Binding of Ethidium bromide to DNA alters its mass and rigidity, and
thereby its mobility.
3. Buffers
Several different buffers have been recommended for electrophoresis of DNA. The
most commonly used buffers are Tris-acetate-EDTA (TAE) and Tris-borate-EDTA(
TBE). The migration rate of DNA fragments in both of these buffers is somewhat
different due to the differences in ionic strength. These buffers provide the ions for
supporting conductivity.
4. Conformation of DNA
DNA with different conformations that has not been cut with a restriction enzyme will
migrate with different speeds. Nicked or open circular DNA will move slowly than
linear and super coiled DNA (slowest to fastest: nicked or open circular, linear, or
super coiled plasmid). Super helical circular, nicked circular and linear DNAs migrate
gels at different rates through agarose gel. The relative nobilities of these three forms
depend on the concentration, type of agarose used to make the gel, applied voltage,
buffer, and the density of super helical twists.
Principle
Agarose gel electrophoresis is the easiest and commonest way of separating and analyzing
DNA were macromolecules are separated based on their electrophoretic mobility. The wide
acceptability of this matrix is its ability to separate a large range i.e. of about 200 bps to
50,000 bps.
1. 10X TAE buffer (pH 8.3) – Tris base - 52.4g

Glacial acetic acid – 11.42ml
EDTA – 7.44g
Volume made upto one litre with water
2. 10X TBE buffer (pH 8.3) – Tris base – 108g

Boric acid – 55g
EDTA – 10.5g
3. DNA loading dye (10X) – 0.25% Bromophenol blue

0.25% Xylene cyanol
30% glycerol in water
4. Ethidium Bromide (10mg/ml)

5. Agarose
91
Procedure
a. Preparation of agarose gel (0.7% gel)
1. Choose the appropriate comb (commonly of 12 slots) and fix into position on the
casting tray adjust so that teeth are 2mm above the casting tray.
2. Take 45 ml of distilled water into a 150 ml flask and add 0.35 g agarose to the
flask and boil to dissolve agar and cool to 45- 55ºC, before adding buffer.
3. Add 5 ml of 10X TAE buffer or TBE buffer
4. Cool the agarose/water mixture to 50ºC and pour on to the leveled plates and
allow it to cool and set least for 30 min.
b. Agarose gel electrophoresis
1. Level off the gel tank using isopropanol. Pour small where of TBE (1X) onto the gel
and remove the comb from the gel. Place the tray in the tank. Pour in the buffer
solution to 2 mm above the gel. Check the level of the tank and adjust if necessary.
2. Add sample loading dye to the samples (DNA). Make appropriate ratio of dye with
samples (1:10 normally). Gently mix.
3. Pipette the samples into the slots taking great care. It helps if these correspond to the
original table. Change the tip between each operation.
4. Replace the lid. Connect the tank to the power supply (noticing the colour codes).
Switch on the power and increase the voltage from 60 volts to 80 volts after 30 mins
and the run will be completed by 1-2 hrs.
5. At the end of the run, the marker band should move 1/2 to 2/3 across the gel. Switch
off the power. Lift out the tray. Photograph the gel before discarding in the waste
disposal unit.
M (1st lane): DNA marker 1, 2, 3 (2nd, 3rd &4th lane):

Genomic DNA of soybean leaves
Note:
1. Prepared EtBr should be kept in amber coloured bottle or store in dark. While
photographing the gel on photodyne under UV light, protect your eyes using UV
goggles.
2. Loading buffer components, Bromophenol blue migrates at a rate equivalent to 200–

400 bp DNA & Xylene cyanol migrates at approximately 4kb equivalence.
92
References
Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular cloning: A Laboratory
manual. 2nd ed. Cold spring harbor laboratory press, cold spring harbor, NY, U.S.A.
93
Agarose gel Electrophoresis of RNA
Principle
RNA is separated using denaturing agarose gel. Guanidine-thyocyanate (GTC) and MOPS(3-
(n-Morpholino)Propanesulfonic Acid).Mean of Platts Singapore (crude oil pricing) can be
used as denaturants. The mobility of RNA is inversely proportional to the log molecular
weights of the denatured RNA species.
Reagents
1. Ethidium Bromide (10mg/ml)

2. 4M GTC solution 50g Guanidine Thiocyanate
2.5 ml 1M Sodium Citrate pH 7.0
0.7 ml β mercapto ethanol
3. 10X TBE buffer (pH 8.3) - Tris base – 108 g

Boric acid – 55 g
EDTA – 10.5 g
4. RNA Loading dye 2 x - 20% formamide

40% formaldehyde
50 mM phosphate buffer
3 mM EDTA
30% 5x Ficoll blue solution
5. 5 x Ficoll blue solution 25% Ficoll 400
0.1% EDTA
0.5% SDS
0.1% Bromophenol blue
6. 20X MOPS (500ml) - 41.9 g MOPS

6.8 g sodium acetate (MW- 136.08)
2.6 g EDTA (MW-372.24)
7. 400ml DEPC water (Adjust pH to 7.0 with NaOH make to 500ml with DEPC-treated
water)
8. 2X RNA Dye - 1X MOPS
50% glycerol
Procedure (GTC as reagent)
a) Weigh 0.36 g of agarose and add 30 ml of 1 x TBE in a flask.

b) Boil it to dissolve agarose and cool to room temperature.
94
c) Add 0.6 ml of 1 M GTC and 3 µl EtBr (lx TBE is prepared with DEPC treated
water).
d) Gel solution is poured into the casting tray with comb in place. Mix RNA samples
with sample buffer and heat at 65°C for 10 min.
e) Remove the comb carefully and place the gel tray in the electrophoresis
apparatus.
f) Pour 1 x TBE just enough to cover the gel and load the samples in the well. Run
the gel at 5V/cm till the dye reaches three fourth of the gel.
Procedure (MOPS as a denaturant)
1. Prepare a 1 % agarose gel in DEPC treated water.

2. Boil and then cool to room temperature.
3. Add MOPS diluted from 20X MOPS buffer stock to 1X and 2 µl ethidium
bromide.
4. To prepare 25 ml agarose by combining 0.25 g agarose, 23 ml DEPC treated water,
1.25 ml DEPC 20X MOPS,0.75 ml 37 %(12.3M) formaldehyde.
5. Sample preparation for loading include 2-5µg of total RNA(adjust the volume of
each RNA sample to 5 µl with water if needed,then add 1.25 µl 20 X MOPS
buffer, 4.5 µl (12.3 M) formaldehyde, 12.5 µl formamide and 2 µl (5X) RNA
loading dye.
6. Incubate at 650 C for 5-10 minutes and load for electrophoresis in 1X MOPS
running buffer and run the gel at 5V/cm till the dye reaches three fourth of the gel.
7. View both the gels under UV trans-illuminator and photograph. When resolved by
electrophoresis the 28S, 18S eukaryotic RNA should exhibit a near 2:1 ratio on
ethidium bromide staining indicating that no significant degradation of RNA has
occurred.
95
Elution of DNA Band from Agarose Gel
Principle
Among the different methods of isolating DNA fragments from agarose gel, electro elution
method is appropriate for purifying larger amounts (>500ng) while elution using phenol
extraction or electrophoresis onto DEAE cellulose paper are useful for smaller scale
purifications. Here is a simple procedure for isolating DNA fragments using low
gelling/melting temperature agarose gels is described. In this method following preparative
gel electrophoresis using low gelling/melting temperature agarose, the band of interest is
removed from the gel. The agarose slice is then melted and subjected to phenol extraction.
Reagents
1. Low gelling agarose
2. 10X TBE buffer (pH 8.3) – Tris base – 108g
3. Buffered phenol Boric acid – 55g
4. BSA (Optimal) Volume EDTA – 10.5g
made upto one litre with water
Procedure
1. Digest DNA sample to completion. Pour, load and electrophorese a 1% low
gelling/melting temperature agarose gel in 1X TBE buffer.
96
2. Stain the gel using EtBr and cut out the desired band with a scalpel
3. Melt the gel slice at 65ºC and add enough TE buffer to decrease agarose
percentage to 0.4% or less
4. Add an equal volume of buffered phenol; mix vigorously for 5 to 10 min; and
centrifuge at 10,000 rpm for 10 min at room temperature.
5. Collect the aqueous phase and set aside. Re-extract phenol phase and interface
with an equal volume of TE buffer. Centrifuge as above.
6. Collect second aqueous phase. If large interface still appears re-extract once more.
7. Combine all the aqueous phase and ethanol precipitate.
Note
1. Use buffered phenol and not phenol: chloroform:isoamyl mixture. A thick white
interface will be visualized after centrifugation.
2. The pellet may be large as it may contain some remnants of agarose
3. Both the quantity of DNA that can be loaded and the quality of resolution are
lesser on a low gelling/melting temperature gel than on a standard agarose gel.
References
Blin. N., Gabain. A. V. and Bujard H. (1975). Isolation of large molecular DNA from
agarose gels for further digestion by restriction enzymes. FEBS Letters, 53: 84-86
97
Restriction digestion of genomic and plasmid DNA
Principle
This is an enzymatic technique which can be used for cleaving DNA molecules at specific
sites, ensuring that all DNA fragments that contain a particular sequence have the same size;
furthermore, each fragment that contains the desired sequence has the sequence located at
exactly the same position within the fragment. The cleavage method makes use of an
important class of DNA-cleaving enzymes isolated primarily from bacteria. These enzymes
are called restriction endonucleases or restriction enzymes, and they are able to cleave DNA
molecules at the positions at which particular short sequences of bases are present.
Reagents
1. Restriction enzymes – (5units/µgDNA)
2. TE buffer
3. Molecular weight marker
Procedure
1. Add the ingredients listed below to a siliconised eppendorf tube in the following order
a. DNA (1-2µg/10µl) to the bottom of the eppendorf tube 10µl
b. Water (added through the sides of the eppendorf tube) 7µl
c. 10X salt buffer to the side of the eppendorf tube 2µl
d. Enzymes (through the side) of the eppendorf tube (5units/µgDNA) 1µl
Total volume = 20µl
98
Mix the ingredients by flicking the eppendorf tubes. Briefly centrifuge all samples to
bring the liquid to the bottom of the tube.
2. Incubate at 37ºC for 2-3 hrs to complete the restriction
3. Unrestricted samples are kept to one side in ice. They contain the appropriate amount
of DNA made up to 20µl using TE buffer in at 20ºC
4. While waiting for the restriction, agarose gel can be prepared
5. Place the eppendorf tubes in water bath at 70-75ºC for 3-4 min and leave in ice until
required.
6. Load samples on agarose gel (0.7 to 0.8%) at 40 mA for 2-3 hrs. Use lambda DNA
digested with Hind III as a molecular weight marker.
Note
The restriction enzyme solutions are 50% glycerol, whilst the maximum level of
glycerol permitted in the restriction samples is 5% (to avoid random restriction).
Thus, for 20µl final volume the permitted total volume for all enzymes is 2µl.
99
PCR-based Cloning of Gene
ArunaTyagi and Kishwar Ali

110012
Introduction
Generally, cloning refers to the isolation/amplification of a DNA fragment or gene of interest
in vitro, its insertion into a vector backbone, and its propagation in live host cells without any
change in the original DNA sequence. Cloning of the gene of interest or a fragment of DNA
is a basic step in molecular biology research. To characterize the function of a particular
DNA sequence, its cloning is the first step. There are two main ways to achieve this: (i)
polymerase chain reaction (PCR) to in vitro amplify it, and (ii) use of restriction enzyme(s) to
cut out the desired DNA fragment followed by inserting the DNA fragment into cloning
vector. The recombinant plasmid thus generated can be propagated/multiplied live in host
cells, e.g., E. coli. PCR is a useful technique to quickly and easily amplify the desired
fragment of DNA, if the sequence information (at least the flanking sequence) is available to
design a pair of primers. Once the DNA fragment has been inserted in a vector, it can be used
to generate a large number of the DNA fragment for further analysis of the gene.
Principle
In molecular cloning, we make use of the fact that the structural unit of DNA is
fundamentally the same in all the living organisms. Therefore, when a segment of DNA is
inserted into a vector containing the functional sequences required for DNA replication, and
the resulting recombinant DNA is introduced into the living cells of a host organism, then the
inserted DNA/gene also gets multiplied along with the replication/multiplication of the
plasmid/vector and division of host cell.
The basic cloning involves following steps:

(1) Choice of host organism and cloning vector,
(2) Preparation of vector DNA,
(3) Preparation of DNA to be cloned
(4) Creation of recombinant DNA,
(5) Introduction of recombinant DNA into host organism,
(6) Selection of organisms containing recombinant DNA,
(7) Screening for clones with desired DNA inserts and biological properties
Choice of host organism and cloning vector

A plasmid is a small extra chromsomal and self replicating circular double-stranded
DNA molecule within a cell that can replicate independently. They are most commonly found
in bacteria as small circular, double-stranded DNA molecules; however, plasmids are
sometimes present in archaea and eukaryotic organisms. Plasmids range in size from a few
thousand base pairs to more than 100 kilobases (kb). Like the host-cell chromosomal
DNA, plasmid DNA is duplicated before every cell division. During cell division, at least one
copy of the plasmid DNA is segregated to each daughter cell, assuring continued propagation
100
of the plasmid through successive generations of the host cell.In nature, plasmids often carry
genes that may benefit the survival of the organism, for example antibiotic resistance.
Plasmids usually are very small and contain only additional genes that may be useful to the
organism under certain situations or particular conditions. Artificial plasmids are widely used
as vectors in molecular cloning which serve the purpose to drive the replication
of recombinant DNA sequences within host organisms.Artificially constructed plasmids are
generally used as vectors in genetic engineering. These plasmids serve as important tools in
genetics and biotechnology research to clone and amplify or express particular genes. A wide
variety of plasmids are commercially available for such uses. A vector ideally has a gene that
confers resistance to particular antibiotic (ampicillin is most frequently used for bacterial
strains), an origin of replication to allow the bacterial cells to replicate the plasmid DNA, and
a suitable site for cloning and a tag gene that can be used to screen for cells containing the
foreign DNA.
Map of pGEM-T vector
Preparation of vector and insert DNA for cloning

Both fragment and vector DNA should be restricted with appropriate restriction enzymes to
generate compatible ends for cloning. Most of the commonly used plasmid vectors have
multiple cloning sites which enable one to use them for directional cloning as well as cloning
of inserts with range of restriction sites on their ends. If a single restriction enzyme is used to
prepare the vector, the vector DNA should be treated with calf intestinal alkaline phosphatase
(CIAP) to remove 5' phosphate groups to prevent re-circularization of the vector during
ligation. The insert DNA can be purified after restriction either by electrophoresis on an
acrylamide or low melting agarose gel and then eluted from the gel.
If DNA for cloning is produced using PCR then cloning differs from traditional
cloning. Typically, a PCR reaction is performed to amplify a DNA fragment or gene of
interest, and then it is joined to the vector via a blunt or single-base overhang ligation prior to
transformation. Taq DNA Polymerase is used to amplify the gene which results in a PCR
product with a single template-independent base addition of an adenine (A) residue to the 3'
end of the PCR product, through the normal action of the polymerase. These "A-tailed"
products are then ligated to a complementary T-tailed vector egp GM-T (having overhang-T
complementary to A) using T4 DNA ligase, followed by transformation. In this case there is
no need to prepare the vector for cloning by using restriction enzymes.High-fidelity DNA
polymerases are also now routinely used to amplify sequences with the PCR product
containing no 3' extensions. The blunt-end fragments are joined to a plasmid vector through a
typical ligation reaction or by the action of an "activated" vector that contains a covalently
attached enzyme, typically Topoisomerse I, which facilitates the vector: insert joining. Some
PCR cloning systems contain engineered "suicide" vectors that include a toxic gene into
which the PCR product must be successfully ligated to allow propagation of the strain that
101
takes up the recombinant molecule during transformation. These vectors for cloning PCR
products are typically sold by suppliers, like NEB, Promega and many others in a ready-to-
use for cloning.
Ligation
Ligation is the joining of two DNA fragments with enzyme ligase to create recombinant
DNA molecules when a foreign DNA fragment is inserted into a plasmid. The ends of DNA
fragments are joined together by the formation of phosphodiester bonds between the 3'-
hydroxyl of one DNA terminus with the 5'-phosphoryl of another.T4 DNA ligase is used for
ligation.
Topoisomerase can also be used for ligation, and the cloning may be done more
rapidly without the need for restriction digest of the vector or insert. In this TOPO
cloning method a linearized vector is activated by attaching topoisomerase I to its ends, and
this "TOPO-activated" vector may then accept a PCR product by ligating to both of the 5'
ends of the PCR product, the topoisomerase is released and a circular vector is formed in the
process.
Before starting the ligation, estimate the concentration of vector and insert DNA by
agarose gel electrophoresis along with molecular weight standards of a known concentration.
Various vector: DNA insert ratios should be tested in order to find out the optimum ratio for
a particular vector and insert. In most cases either a 1:1 or a 1:3 molar ratio of vector: insert
works well.
The factors effecting Ligation

DNA concentration: The concentration of DNA affects the rate of ligation, Ligation involves
joining up the ends of a DNA with other ends, however, if the ends of plasmid DNA are
compatible ( when restricted with single restriction enzymes), can circularize by joining its
own ends.Plasmid vector DNA needs to be dephosphorylated to avoid a high background of
recircularized vector DNA with no insert. This is commonly done using calf-intestinal
alkaline phosphatase (CIAP) which removes the phosphate group from the 5′ end of digested
DNA. At high DNA concentration, there is a greater chance of forming intermolecular
ligation, while at a lower DNA concentration, intramolecular reaction is more likely to take
place. The relative concentration of the DNA fragments, as well as their length, are also
factors that can affect whether intermolecular or intramolecular reactions are favored. The
molar ratio of insert to vector usually used is 3:1. Very high ratio may produce multiple
inserts. The ratio may be adjusted depending on the size of the insert.
I. Higher the concentration of ligase reaction mixture, the faster the rate of ligation.
Blunt-end ligation is much less efficient than sticky end ligation, Threfore, minimal
volume reaction mixture should be prepared to achieve a higher concentration. High
DNA ligase concentration may be used in conjunction with PEG for a faster ligation.
II. The optimum temperature for DNA ligase activity is 37 °C and melting
temperature (Tm) of the DNA ends to be ligated is dependent on length and base
composition of the DNA overhangs i.e. greater the number of G and C, higher the Tm.
For the efficient ligation, the ends should be stably annealed.The Tm of the DNA ends
is generally much lower than 37°C. The optimal temperature for ligation should be used
102
as per the instruction provided by the protocols supplied with ligase. Most of protocols
suggest 12-16°C, room temperature, or 4°C.
Competent Cell
E. coli cells can incorporate foreign DNA if their cell walls are altered so that DNA can pass
through it. Such cells are said to be "competent”. Cells are made competent by a process that
uses calcium chloride and heat shock. Cells that are undergoing very rapid growth are made
competent more easily than cells in other stages of growth. ln CaCl2 method, the competency
can be obtained by creating pores in bacterial cells by suspending them in a solution
containing high concentration of calcium.
The choice of correct type of competent cells is very much important for blue white
screening. The plasmid to be used for cloning must contain the lacZα, eg pUC19,
pGEM,pBluescript etc. The E. coli cell to be used for competent cells should contain the
mutant lacZ gene with deleted sequence (i.e. lacZΔM15). The commonly used cells with such
genotype are JM 107, JM109, DH5α and XL1-Blue.
Transformation
Transformation is a process of horizontal gene transfer by which some bacteria take up

foreign genetic material (naked DNA) from the environment. It was first reported in
Streptococcus pneumoniae by Griffith in 1928 and demonstrated by Avery et al in 1944. The
process of gene transfer by transformation does not require a living donor cell but only
requires the presence of persistent DNA in the environment. The prerequisite for bacteria to
undergo transformation is its ability to take up free, extracellular genetic material. Such
bacteria are termed as competent cells. The exogenous genetic material may co-exist as a
plasmid with chromosomal DNA.There is two methods to transform E. coli cells with
plasmid DNA - chemical transformation and electroporation. For chemical transformation,
cells are grown to mid-log phase, harvested and treated with divalent cations such as CaCl2.
To chemically transform cells, competent cells are mixed with the DNA, on ice, followed by
a brief heat shockat 42oC. Then, cells are incubated with rich medium and allowed to express
the antibiotic resistant gene for 30-60 minutes prior to plating.
For electroporation, cells are also grown to mid-log phase and then washed
extensively with water to eliminate all salts. Usually, glycerol is added to the water to a final
concentration of 10% so that the cells can be stored frozen and saved for future experiments.
To electroporate DNA into cells, washed E. coli cells are mixed with the DNA to be
transformed and then pipetted into a plastic cuvette containing electrodes. A short electric
pulse, about 2400 volts/cm, is applied to the cells causing smalls holes in the membrane
through which the DNA enters. The cells are then incubated with broth as above before
plating.
The blue-white screening

The blue-white screening technique allows for the rapid and convenient detection of
recombinant bacteria in vector-based molecular cloning experiments. DNA of interest is
ligated into a vector. The vector is then inserted into competent host cells viable for
transformation, which are then grown in the presence of X-galand IPTG. Cells transformed
with vectors containing recombinant DNA will produce white colonies; cells transformed
with non-recombinant plasmids (i.e. only the vector) grow into blue colonies.
103
The method is based on the principle of α-complementation of the β-
galactosidase gene. This phenomenon of α-complementation was first demonstrated in work
done by Ullmann ctal (1967). β-galactosidase is a protein encoded by the lacZ gene of
the lac operon, and it exists as a homotetramer in its active state. However, a mutant β-
galactosidase derived from the M15 strain of E. coli has its N-terminal residues 11—41
deleted and this mutant, the ω-peptide, is unable to form a tetramer and is inactive. This
mutant form of protein may return fully to its active tetramer state in the presence of an N-
terminal fragment of the protein, the α-peptide. The rescue of function of the mutant β-
galactosidase by the α-peptide is called α-complementation.
In this method of screening, the host E. coli strain carries the lacZ deletion mutant
(lacZΔM15} which contains the ω-peptide, while the plasmids used carry the lacZα sequence
which encodes the first 59 residues of β-galactosidase, the α-peptide. Neither of them are
functional alone. However, when the two peptides are expressed together, as when a plasmid
containing the lacZα sequence is transformed into a lacZΔM15 cells, they form a
functional β-galactosidase enzyme. The blue/white screening method works by disrupting
this α-complementation process. The plasmid carries within the lacZα sequence an
internal multiple cloning site (MCS). This MCS within the lacZα sequence can be cut by
restriction enzymes so that the foreign DNA may be inserted within the lacZα gene, thereby
disrupting the gene that produces α-peptide. Consequently, in cells containing the plasmid
with an insert, no functional β-galactosidase may be formed. The presence of an active β-
galactosidase can be detected by X-gal, (a colourless analog of lactose) that may be cleaved
by β-galactosidase to form 5-bromo-4-chloro-indoxyl, which then spontaneously dimerizes
and oxidizes to form a bright blue insoluble pigment 5,5'-dibromo-4,4'-dichloro-indigo. This
results in a characteristic blue colour in cells containing a functional β-galactosidase. Blue
colonies therefore show that they may contain a vector with an uninterrupted lacZα (therefore
no insert), while white colonies, where X-gal is not hydrolyzed, indicate the presence of an
insert in lacZα which disrupts the formation of an active β-galactosidase. Isopropyl β-D-1-
thiogalactopyranoside (IPTG) is also used with X-gal which functions as the inducer of
the lac operon, to enhance the expression of LacZ.
Preparation of vector and insert DNA for cloning

Restriction of plasmid DNA (Vector) with appropriate enzyme
Plasmid DNA - 5 µg
10X Buffer (for appropriate restriction enzyme) - 5 µl
1 mg/ml acetylated BSA (Optimal) - 5 µl
Appropriate restriction enzyme - 5 to 25 units
Deionized water - to final volume 50µl
Incubate at an appropriate temperature for 1-3 hr. Run an aliquot on a 1% agarose gel to
check completion of restriction.
(b) CIAP treatment of vector for dephosphorylation

1. CIAP 10X Buffer
Tris-HC1, pH 9.0 - 500 mM
MgC12 - 10 mM
ZnCl2 - 1 mM
104
2. Phenol : chloroform : isoamylalcohol (25 : 24 : 1)
3. Chloroform : isoamylalcohol (24 : 1)
4. Sodium acetate 3M (pH 5.2)
5. Ethanol 100% and 70%
6. 0.5 M EDTA
7. 7.5 M Ammonium acetate
Protocol
1. To the vector DNA restriction mix add the following directly
CIAP 10X buffer - 10 µl
CIAP - 0.01U/pmol ends* nuclease free water
- to final volume of 100 µl
* e.g. 1 µg of a 1 Kb DNA fragment = 1.52 pmol DNA, or 3 pmol ends.
CIAP may be diluted in 10x CIAP buffer immediately before use and discard unused
diluted enzyme.
2. Incubate using one of the following conditions depending upon the type of ends present.
(i) For 5' protruding ends: Incubate at 37°C for 30 min, then at 56°C for 15 min. Add
another 0.014 unit CIAP/pmol ends and repeat incubations at both the temperatures.
(ii) For blunt or 5' recessed ends: Incubate for 15 min at 37°C then for 15 min at 56°C.
Add another 0.01 Unit CIAP/pmol ends and repeat incubation at both the
temperatures.
3. Stop the reaction by adding 2.0 µl of 0.5 M EDTA and heating at 65°for 20 min.
4. Extract the DNA by adding an equal volume of phenol: chloroform: isoamyl alcohol (25
: 24 : 1) vortex for 1 min and centrifuge at 12,000 x g for 5 min.
5. Carefully remove the top aqueous phase to a fresh tube (To increase recovery the
organic phase can be back-extracted with a small volume of TE buffer).
6. Add an equal volume of chloroform: isoamyl alcohol (24: 1) vortex and centrifuge at
12,000 x g for 2 min. Remove aqueous phase to a fresh tube.
7. Precipitate the DNA by adding 1/10 volume of 3 M sodium acetate (pH 5.5) and 2
volumes of 100% ethanol. Place the sample on ice or at -20°C for 30 min.
8. Pellet the DNA by centrifugation at 12,000 x g for 5 min, wash with 70% ethanol, air
dry and dissolve in appropriate amount of TE Buffer.
(c) Preparation of insert DNA

a. Restrict the genomic DNA with suitable enzyme and separate on an agarose gel.
b. Elute the DNA from the agarose gel.
c. Check an aliquot of the eluted DNA on the gel.
(d) Ligation of plasmid vector and insert
T4 DNA ligase 10x Buffer
Tris-HC1, pH 7.8 - 300 mM
MgC12 - 100 mM
DTT - 100 mM
ATP - 10 mM
105
Procedure
1. Using the appropriate vector: insert ratio set up the following reaction.
Generally 50-200 ng vector DNA is used in a typical ligation reaction.
Vector DNA - 100 ng
Insert DNA - 50 ng
10x Ligase buffer - 1 µl
T4 DNA ligase (Weiss unit) - 1 unit
Nuclease free water to final volume - 10 µl
2. Incubate the reaction mix either at 22°C for 4 hr or at 4°C for 16 hr (overnight) for
blunt ends or at 22°C for 3 hr or 4°C for 16 hr (overnight) for protruding ends.
3. After the ligation reaction is over transform appropriate amount of ligated DNA into
competent cells of an appropriate host strain.
Preparation of competent cells

Solutions/Materials required
1. LB Broth
Bactotryptone - 10 g
NaCl - 10 g
Yeast extract - 5g
DDW - 1 litre
Adjust pH to 7.5 with NaOH and autoclave.
2. LB agar plates Add 15.0 g/lit agar in LB broth, sterilize, cool and add proper
antibiotic and pour in petriplates.
3. MgCl2, 0.1 M
4. CaCl2, 0.1 M
5. 50% glycerol/50 mM CaC12 solution
6. SOC Medium –
2% tryptone
0.5% yeast extract
10 mMNaCl
20 mM glucose (dextrose)
10 mM MgC12
10 mM MgSO4
MgC12, MgSO4 and glucose are prepared separately and added to the broth.
Procedure
1. Inoculate 10 ml LB broth with a loopful of an appropriate strain of E. coli cells from a
single colony and grow at 37°C with shaking at 200 rpm overnight.
2. Subculture 5 ml of the overnight grown culture with 500 ml of LB broth in a 2 litre
flask.
3. Grow with shaking at 37°C to an OD600 of 0.4/0.5 as measured by a
spectrophotometer (usually 2-3 hr).
106
4. Chill the cells on ice. Pour the culture into 250 ml plastic centrifuge bottles and spin
at 2500 x g for 5 min. decant the supernatant.
5. Resuspend the pellets in 100 ml of cold (0-4°C) 0.1 M MgCl2 (1/5th of the original
culture volume) and transfer the cell suspension to two 50 ml oakridge tubes. The
cells must be kept at 4°C.
6. Incubate the cells on ice for 5 min.
7. Centrifuge the cells at 2,500 x g for 5 min at 4°C. Decant the supernatant.
8. Wash the cell pellets with ice cold 0.1 M CaC12 (do not vortex). Centrifuge at 2,500 x
g for 5 min. at 4°C and decant supernatant.
9. Suspend each pellet in 7 ml of ice cold 0.1 M CaCl2.
10. Incubate the cells on ice overnight.
11. Add 3 ml of ice cold 50% glycerol/50 mM CaC12 to each tube and mix gently.
12. Aliquot 0.5 ml of cells into prechilled tubes and quick freeze in liquid nitrogen and
store the frozen cells at -80°C.
Transformation
1. Rapidly thaw competent cells (100 µl aliquot) by warming between hands.
2. Add 10 ng of DNA (5-10 µl volume of ligation mix) into microcentrifuge tube and
place on ice. Swirl with help of tip of and place on ice for 30 min.
3. Give heat shock to the cells, place tubes for 45-60 second sat 42°C in water bath and
quickly chill the tubes for 1-2 min.
4. Add 1 ml SOC/LB medium to each tube.
5. Incubate for 45 Minutes at 37°C on shaker at150 rpm.
6. Plate different aliquots on LB plates containing appropriate antibiotic,50µlX-gal of 50
mg /ml X-gal and 100 µl of o.1M and incubate for overnight at 37°C.
7. Store remaining cells at 4°C for subsequent plating. Transformation efficiencies range
from 106 to 108transformants/µg DNA.
Final concentrations of antibiotics in the plate should be 100 µg /m1 for ampicillin, 12.5
µg/m1 for tetracycline, 30 µg/ml for kanamyc in or 20 µg/m1 for chloramphenicol.
(iii) Blue/white color screening

The colonies carrying recombinants appears white and the colonies carrying non-
recombinants appears blue. The white colonies identified and transferred to another LB
plates containing appropriate antibiotic and grown overnight at at 37°C for further study.
107
PCR amplification and cloning of the gene of interest
1% agarose gel showing plant DNA 1% agarose gel showing Blue and white colonies after ligation and
PCR amplified product transformation on LB plates containing
apmiciline, IPTG and X-GAL
Plasmid vector
Plasmid vector
Gene of interest released

after restriction
Gene of interest after cloning in plasmid

vector and restriction On 1% agarose gel
Notes
1. An illustrative example for conversion of molar ratios to mass ratios is given for a 3.0 kb
vector and 500 bp insert.
ng of vector × kb size of insert insert
×molar ratio of = ng insert
kb size of vector vector
e.g. Amount of 500 bp insert DNA needs to be added to 100 ng of 3.0 kb vector in a ligation
reaction with vector insert ratio of 1:3.
100 ng of vector × 0.5 kb of insert 3

×molar ratio of = 50 ng insert
3.0 kb of vector 1
Calculation of transformation efficiency

108
For each batch of competent cells prepared a control transformation should be done with a
known quantity of supercoiled plasmid.
For example: A 100 µl competent cells is transformed with 1 ng of a supercoiled plasmid

vector DNA. Ten microlitres of transformation reaction (0.1 ng total DNA) is added to 990
µl SOC medium (1: 100 dilution). Of that volume a 100 µl aliquot is plated (1: 1000 final
dilution) and one hundred colonies were obtained on the plate. Then,
3 3
100 Cfu x 10 ng x 10 9
Transformation efficiency (cfu/µg) = × 1 x 10
0.1 ng of plasmid 1 µg
References
Bernard R. Glick, Jack J. Pasternak (2010) Molecular Biotechnology, Fourth edition,

American Society for Microbiology.
Brown T (2006). Gene cloning and DNA analysis: an introduction. Cambridge, MA:
Blackwell Pub. ISBN 978-1-4051-1121-8.
Dagert, M.; Ehrlich, S. (1979). "Prolonged incubation in calcium chloride improves the
competence of Escherichia coli cells". Gene. 6 (1): 23–28
Dugaiczyk A, Boyer HW, Goodman HM (July 25, 1975). "Ligation of EcoRI endonuclease-
generated DNA fragments into linear and circular structures". Journal of Molecular
Biology. 96 (1): 171–84
Langley, K. E.; Villarejo, M. R.; Fowler, A. V.; Zamenhof, P. J.; Zabin, I. (1975). Molecular
basis of beta-galactosidase alpha-complementation. PNAS (USA). 72 (4): 1254–1257.
Lederberg J (Feb 1994). The transformation of genetics by DNA: an anniversary celebration
of Avery, MacLeod and McCarty (1944) . Genetics. 136 (2): 423–6.
Sambrook, J., Fritsch, F.F. and Maniatis, T. (1989). Molecular Cloning- A Laboratory
Manual. Cold Spring Harbor Laboratory Press.
Sandy Primrose and Richard Twyman, Principles of gene manipulation and Genomics,
Seventh edition, Blackbell publishing, 2006.
Tom Maniatis, E. F. Fritsch, Joseph Sambrook, Molecular cloning-a laboratory manual, 7th
edition, Cold Spring Harbor Laboratory, 1982.
Ullmann, A.; Jacob, F.; Monod, J. (1967). "Characterization by in vitro complementation of a
peptide corresponding to an operator-proximal segment of the beta-galactosidase structural
gene of Escherichia coli". Journal of Molecular Biology. 24 (2): 339–343.
109
Preparation of Vector and Insert DNA for Cloning
Add DNA restriction mix to the vector and incubate
Stop the reaction by adding 2.0 µl of 0.5 M EDTA and heating at 65°for 20 min.
Add an equal volume of phenol: chloroform: isoamyl alcohol (25 : 24 : 1)
Vortex for 1 min and centrifuge at 12,000 x g for 5 min.
Take top aqueous phase to a fresh tube
Add an equal volume of chloroform: isoamyl alcohol (24: 1)
Vortex and centrifuge at 12,000 x g for 2 min.
Remove aqueous phase to a fresh tube.
Add 1/10 volume of 3 M sodium acetate and 2 volumes of 100% ethanol.
Place the sample on ice or at -20°C for 30 min.
Centrifugation at 12,000 x g for 5 min
Wash with 70% ethanol, air dry and dissolve in appropriate amount of TE buffer.
110
Preparation of Competent Cells
Inoculate 10 ml LB broth with E. coli cells and incubate at 37°C with shaking at 200 rpm for
overnight
Sub-culture 5 ml with 500 ml of LB broth in a 2 liter flask
Grow with shaking at 37°C to an OD600 of 0.4/0.5

(Usually 2-3 hr)
Chill the cells on ice. Pour the culture into 250 ml plastic centrifuge bottles and spin at 2500
x g for 5 min, decant the supernatant
Resuspend the pellets in 100 ml of cold (0-4°C) 0.1 M MgCl2 (1/5th of the original culture
volume)
Transfer the cell suspension to two 50 ml oakridge tubes.
Incubate the cells on ice for 5 min.
Centrifuge the cells at 2,500 x g for 5 min at 4°C. Decant the supernatant
Wash the cell pellets with ice cold 0.1 M CaC12 (do not vortex)
Centrifuge at 2,500 x g for 5 min. at 4°C and decant supernatant
Suspend each pellet in 7 ml of ice cold 0.1 M CaCl2
Incubate the cells on ice overnight
Add 3 ml of ice cold 50% glycerol/50 mM CaC12 to each tube and mix gently.
Aliquot 0.5 ml of cells into prechilled tubes and quick freeze in liquid nitrogen and store the
frozen cells at -80°C.
111
Isolation of Genomic DNA from Plant
Suresh Kumar and Archana Singh
Division of Biochemistry, ICAR-Indian Agricultural Research Institute, New Delhi-110012
Introduction
The extraction of DNA from plants is the starting point for genotypie analysis.The approach
to preparation of DNA from plants is determined by the species, the type of tissue or sample
available and the analysis required on the DNA. For example, extraction of DNA from the
leaf of a cereal for analysis using a robust test may require a very different technique from
that required for isolation of DNA from the bark of a tree for ampliﬁed fragment length
polymorphism (AFLP) analysis.DNA extraction from plant tissues, unlike DNA isolation
from mammalian tissues, remains difficult due to the presence of a rigid cell wall
surrounding the plant cells. DNA extraction from plant tissue can vary depending on the
material used. Essentially any mechanical means of breaking down the cell wall and
membranes to allow access to nuclear material, without its degradation is required. To check
the quality of the extracted DNA, a sample is run on an agarose gel, stained with ethidium
bromide, and visualized under UV light.
Principle
Extraction of nucleic acids from biological sample requires cell lysis, inactivation of cellular
nucleases and separation of the desired nucleic acid from cellular debris. The basic steps
involved in all the genomic DNA isolation methods are removal of cell wall and cell
membranes debris, proteins, lipids, carbohydrates and RNAs without affecting the integrity
of the genomic DNA. G rinding of the tissue sample in liquid nitrogen (at 197 C) makes it
brittle, and prevents endogenous nuclease activity. Solution containing detergents to
solubilise cell membranes, and strong chaotropic salts to inactivate intracellular enzymes is
used to provide the medium in which the extraction process proceeds. Cetyl-trimethyl
Ammonium Bromide (CTAB) is a cationic detergent which forms an insoluble complex
with nucleic acids in a low-salt environment (< 0.5 M NaCl). Under these conditions,
polysaccharides, phenolic compounds and other contaminants remain in the supernatant and
can be washed away. The DNA complex is then solubilised by raising salt concentration and
precipitated with ethanol or isopropanol.
Homogenised sample is first treated with extraction buffer containing EDTA, Tris-HCl
and CTAB. EDTA chelates magnesium ions, which is an essential cofactor for DNase
enzymes (responsible for degrading DNA), thus it minimises the activity of cellular DNase
and protects DNA from degradation. Tris-HCl gives the solution a pH buffering capacity (as
either low or high pH damages DNA). Solvent extraction process is often used to eliminate
contaminants from the nucleic acid. For example, a combination of phenol and chloroform is
used to remove proteins. Under low salt concentration (< 0.5 M NaCl), the contaminants of
nucleic acid do not precipitate and can be removed by extraction of the aqueous solution
with chloroform. The chloroform denatures proteins and facilitates separation of the aqueous
and organic phases. Normally, the aqueous phase forms the upper phase. If needed, the
extraction with chloroform is performed two or three times in order to completely remove
the impurities from the aqueous layer. Once the nucleic acid complex has been purified, the
last step of the procedure, i.e. precipitation, can be accomplished using isopropanol or
ethanol to concentrate nucleic acids.
112
CTAB method
CTAB method, first developed by Murray and Thompson in (1980), eliminates
polysaccharides and polyphenolic compounds, particularly from plant materials, and
centrifugation removes cell wall debris, proteins, lipids carbohydrates and RNA without
affecting the integrity of DNA.
One of the most widely used isolation protocol involves the use of a nonionic detergent cetyl
trimethyl ammonium bromide (CTAB) which complexes with carbohydrates and can be
phenol extracted. It is very simple and can be used for isolating good quality DNA for
various applications from a wide variety of plant genera.
Reagents
1. Extraction buffer
1.4 M NaC1
2.0% CTAB
100 mM Tris-HC1, pH 8.0
20 mM EDTA
0.2 % β-mercaptoethanol (add just before use)
2. Chloroform: Isoamyl alcohol mixture (24 : 1, v/v)
3. 3 M Sodium acetate, pH 5.2
4. TE Buffer
10 mM Tris-C1, pH 8.0
1 mM EDTA
5. Isopropanol
6. RNase A (10 mg/ml)
Dissolve RNase A in 10 mM Tris-C1 (pH 7.5) containing 15 mM NaCI. Heatin a
boiling water bath for 10 15 min. Cool to room temperature and store at 20°C
in small aliquots.
7. Phenol saturated with TE.
Protocol
(a) Extraction of DNA
1. Grind 5 g of plant tissue in liquid nitrogen to a fine powder using pre-chilled mortar
and pestle.
2. Transfer the powder to a 250 ml conical flask containing 50 ml of pre-warmed
extraction buffer. Use a spatula to disperse the material completely.
3. Incubate the mixture at 60°C for 30 min with occasional mixing by gentle swirling.
4. Add an equal volume of chloroform-isoamyl alcohol mixture, mix the contents gently
followed by centrifugation at 10,000 rpm for 10 min in 50 ml oakridge tubes.
5. Collect the upper aqueous phase and repeat the above step till no white interface is
visible.
6. To the aqueous phase add 0.6 volume of isopropanol and mix gently and leave at
20°C for 1 h.
7. Pellet the DNA at 10,000 rpm and wash it with 70% ethanol.
8. Air-dry the pellet and dissolve in appropriate quantity of TE.
113
(b) Purification of DNA
1. To the DNA solution add 20 µg/m1 DNase-free RNase and incubate at 37°C for 1 h.
2. Add 0.1 volume of 3 M sodium acetate pH 5.2.
3. Add an equal volume of phenol: chloroform mixture (1:1) and mix well by swirling.
Centrifuge and collect the aqueous phase.
4. Repeat the above extraction step till no white interface is seen.
5. Precipitate the DNA by adding 2 volumes of absolute ethanol. Recover the DNA by
centrifugation, wash it with 70% ethanol, air-dry and dissolve in TE buffer.
The genomic DNA isolated by this method can be used directly for other downstream
processes like PCR, AFLP, restriction digestion, etc. However, RNase treatment before
restriction digestion would be preferable; for other analysis, it can be used without RNase
treatment.
Note: (i) Two major factors that affect quality/integrity of DNA are the presence of nucleases
and mechanical shearing of DNA during isolation steps.
(ii) To reduce endogenous nuclease activity, the plant tissue should be kept frozen in
liquid nitrogen and grinding should be performed in presence of liquid Nitrogem.
The ground tissue must thaw in the extraction buffer containing a detergent and
EDTA.
(iii) Mechanical shearing can be minimized by avoiding rough handling of the lysate and
subsequently of the extracted genomic DNA.
References
Dellaporta SL, Wood J and Hicks JB (1983) Plant DNA mini preparations: Version II. Plant
Mol. Biol. Rep., 1: 19 21.
114
MiniPrep Isolation of Genomic DNA from Plants
Introduction
For high-throughput genotyping and such other analysis, the genomic DNA isolated from
plant tissues must be of sufficient quality to generate and score the data. For screening of a
larger population of plants, isolation of genomic DNA using the above-mentioned CTAB
method of DNA isolation would be very time-consuming and combursome task. Most of the
DNA isolation methods are lenthy, require costly chemicals, equipment and liquid Nitrogen.
Therefore, the DNA isolation method should be fast and simple.
Reagents
1. Extraction buffer
50 mM Tris-C1 (pH 8.0)
100 mM EDTA (pH 8.0)
150 mM NaC1 1.8% SDS
1.5% mixture of PVP and PVPP
2. Phenol: Chloroform: Isoamyl alcohol mix (25: 24: 1)
3. Ethanol
4. TE Buffer
Protocol
1. Take 20 mg of plant tissue; add 100 µl of extraction buffer. Massacreate using sterile
pipette tip.
2. Incubate tubes at 65°C for 30 min with frequent inversion of tubes.
3. Add 100 µl phenol: chloroform: isoamyl alcohol mixture. Mix gently, but thoroughly for
2 min to form an emulsion.
4. Centrifuge at 10,000 rpm for 5 min at 4°C.
5. Remove top aqueous phase to a fresh tube, and add 2 volume of chilled ethanol. Mix by
inverting the tube. Incubate at 20°C for 5 min, and centrifuge at 10,000 rpm for 5 min at
4°C. Drain off the supernatant.
6. Dry the pellet, and dissolve the DNA pellet in 50 µl TE buffer.
References
Murray, M.G. and Thompson, W.F. (1980). Rapid isolation of high molecular weight plant
DNA. Nucl. Acid Res., 8: 4321 4325.
115
Qualitative and Quantitative Analyses of the DNA
Agarose Gel Electrophoresis
Introduction
Agarose gel electrophoresis is the easiest and commonest way of separating and analyzing
DNA where macromolecules are separated based on their electrophoretic mobility. The wide
acceptability of this matrix is its ability to separate a large range i.e. of about 200 bps to
50,000 bps.
Reagents
1. 10 TBE
Tris base - 108 g
Boric acid - 55 g
0.5 M EDTA pH 8.0 - 40 ml
Water - 1 litre
2. 10 X gel loading dye
Glycerol - 5 ml
10 TBE - 1 ml
Bromophenol blue (Saturated) - 1 ml
Xylene cyanol 10% - 1 ml
Mix well, autoclave and store in aliquots
3. 10 mg/ ml Ethidium bromide in sterile water.
Protocol
1. Cast an agarose gel (0.8%) in 1 TAE

2. Load DNA sample (2 5 µl) after adding 1 µl of 6 loading dye.
3. Also load a known volume of λ HindIII DNA as DNA marker in the adjacent well.
4. Run the gel at 50 V for 1 hr.
5. Stain the gel with ethidium bromide (0.5 µg/ml) for 10 min, wash with distilled water
and visualize under UV light.
6. Presence of a single intact band above the upper most band of the marker DNA
indicates high-molecular weight of the isolated genomic DNA.
Figure: Qualitative analysis of the isolated genomic DNA by

agarose gel (0.8%) electrophoresis.
116
Precautions
1. Plant material should not be subjected to frequent freezing thawing.
2. The DNA pellet should not be over-dried, as an over-dried DNA pellet becomes difficult
to be dissolved.
3. All mixing step should be performed gently to avoid mechanical shearing of isolated
genomic DNA.
117
Quantitative analysis of the DNA by Spectrophotometry
Introduction
One of the more commonly used practices to quantitate DNA is the use
of spectrophotometer. A is able to determine the average concentrations of the DNA present
in a solution, as well its purity.
Principle
Spectrophotometric analysis is based on the principles that nucleic acids absorb ultraviolet
(UV) light in a specific pattern. In the case of DNA and RNA, a sample is exposed to
ultraviolet light at a wavelength of 260 nanometres (nm) and a photo-detector measures the
light that passes through the sample. Some of the ultraviolet light may pass through and some
may be absorbed by the DNA / RNA. The more light absorbed by the sample, the higher the
nucleic acid concentration in the sample. The resulting effect is that less light will strike
the photodetector and this will produce a higher optical density (OD)
Using the Beer-Lambert Law, it is possible to relate the amount of light absorbed to the
concentration of the absorbing molecule. At a wavelength of 260 nm, the average extinction
coefficient for double-stranded DNA is 0.020 (μg/ml)−1 cm−1, for single-stranded DNA it is
0.027 (μg/ml)−1 cm−1, for single-stranded RNA it is 0.025 (μg/ml)−1 cm−1 and for short single-
stranded oligonucleotides it is dependent on the length and base composition. Thus,
an Absorbance (A) of 1 corresponds to a concentration of 50 μg/ml for double-stranded
DNA.
Calculations
The optical density is generated from the equation as given below:
Optical Density= Log (Intensity of Incident Light / Intensity of
Transmitted Light)
In practical terms, a sample that contains no DNA or RNA should not
absorb any of the ultraviolet light and therefore produce an OD of 0
Optical Density=Log (100/100)=0
The following absorbance units to nucleic acid concentration conversion factors are used
to convert OD to concentration of unknown nucleic acid samples:
A260 dsDNA = 50 µg/ml
A260 ssDNA = 33 µg/ml
A260 ssRNA = 40 µg/ml
Conversion Factors
When using a 10 mm path length, simply multiply the OD by the conversion factor to
determine the concentration. Example, a 2.0 OD dsDNA sample corresponds to a sample
with a 100 ug/ml concentration.
When using a path length that is shorter than 10 mm, the resultant OD will be reduced by a
factor of 10/path length. Using the example above with a 3 mm path length, the OD for the
118
100 ug/ml sample would be reduced to 0.6. To normalize the concentration to a 10mm
equivalent, the following is done:
0.6 OD X (10/3) * 50 ug/ml=100 ug/ml
Most spectrophotometers allow selection of the nucleic acid type and path length such that
resultant concentration is normalized to the 10 mm path length which is based on the
principles of Beer's law.
Sample Purity (260:280 / 260:230 Ratio)

It is common for nucleic acid samples to be contaminated with other molecules (i.e. proteins,
organic compounds, other). The secondary benefit of using spectrophotometric analysis for
nucleic acid quantitation is the ability to determine sample purity using the 260 nm:280 nm
calculation. The ratio of the absorbance at 260 and 280 nm (A260/280) is used to assess the
purity of nucleic acids. For pure DNA, A260/280 is widely considered ~1.8 but has been argued
to translate - due to numeric errors in the original Warburg paper - into a mix of 60% protein
and 40% DNA.[5] The ratio for pure RNA A260/280 is ~2.0. These ratios are commonly used to
assess the amount of protein contamination that is left from the nucleic acid isolation process
since proteins absorb at 280 nm. The ratio of absorbance at 260 nm vs 280 nm is commonly
used to assess DNA contamination of protein solutions, since proteins (in particular, the
aromatic amino acids) absorb light at 280 nm. The reverse, however, is not true — it takes a
relatively large amount of protein contamination to significantly affect the 260:280 ratio in a
nucleic acid solution. Spectrophotometry is a technique to judge the quality of DNA from
the ratio of the OD values recorded at 260 and 280 nm. The A 260/A280 ratio around 1.8-1.9
indicates best quality DNA. Where A280 is the absorbance wavelength for protein. A
conversion factor of 50 is used to convert OD concentration in µg/ml.
Relationships
A230 ≤ 0.10 1OD = 50µg ds DNA
A230/A260 ≤ 0.45 1OD = 37µg ss DNA
[To determine DNA or RNA concentration by measuring the A260 value. Note that
absorbance measurements cannot discriminate between DNA and RNA]
References
Sambrook & Russell (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold
Spring Harbor Laboratory Press. ISBN 978-0-87969-577-4.
Glasel J. (1995). "Validity of nucleic acid purities monitored by 260/280 absorbance

ratios". BioTechniques. 18 (1): 62–63. PMID 7702855
119
Extraction of DNA
Grind about 1 5 g of plant tissues using liquid Nitrogen
Transfer the powdered tissues in a conical flask, and add pre-warm extraction buffer
Incubate at 60oC for 30 min, and mix occasionally
Add equal volume of chloroform-isoamyl alcohol mixture
Centrifuge at 10,000 rpm for 10 min.
Collect the upper aqueous phase, repeat this step 3 times
Add 0.6 volume of isopropanol, and mix gently by inverting the tube
Incubate at 20oC for 1 h
Centrifuge at 10,000 rpm, and wash the pellet with 70% ethanol
Air-dry the pellet, and dissolve in appropriate quantity of TE buffer
MiniPrep Protocol for Isolation of DNA
Take 20 mg of tissue, add 100 µl of extraction buffer, and crush the tissue
using pipette tip
Incubate the tube at 65°C for 30 min with frequent inversions.
Add 100 µl of phenol: chloroform: isoamyl alcohol mixture, and mix gently
Centrifuge at 10,000 rpm for 5 min at 4°C.
Take the top aqueous phase to a fresh tube
Add 2 volume of chilled ethanol, mix by inverting the tube
Incubate at 20°C for 5 min, and centrifuge at 10,000 rpm for 5 min at 4°C.
Discard the supernatant, air-dry the DNA pellet and dissolve in 50 µl TE
120
Purification of DNA
Add 20 µm/ml DNase-free RNase to the isolated DNA
Incubate at 37oC for 1 h.
Add 0.1 volume of 3M sodium acetate (pH 5.2)
Add equal volume of phenol: chloroform (1:1) and mix well
Centrifuge and collect the aqueous phage, repeat this step three times
Precipitate the DNA by adding 2 volume of absolute ethanol
Centrifuge it to precipitate DNA
Add 70% ethanol, air dry the pellet, and dissolve the pellet in TE buffer
Evaluate the quality of genomic DNA by agarose gel electrophoresis
121
Isolation of RNA and Synthesis of cDNA
Archana Singh
110012
Introduction
RNA is found in the nucleus, cytoplasm and mitochondria of eukaryotic plant cells. Total
cytoplasmic RNA consists of ribosomal RNA (rRNA) transfer RNA (tRNA), messenger
RNA (mRNA) and other small species of RNA. Only 1-3% of the total RNA in eukaryotic
cells is mRNA, majority of the total RNA consists of rRNA. Construction of cDNA libraries
requires highly purified mRNA for efficient cDNA synthesis. RNA is notoriously susceptible
to degradation and special care is required for its isolation. Four important steps as given
below are important to take care for the successful isolation of intact plant RNA by any
procedure of its choice.
Steps of RNA isolation

1. Effective disruption of cells or tissues
2. Denaturation of nucleo-protein complexes
3. Inactivation of endogenous ribonuclecase activity
4. Purification of RNA from contaminating DNA and protein
[To avoid RNase contamination during the process of RNA isolation, chemical- diethyl pyro-
carbonate (DEPC). This chemical inactivates ribonuclease by covalent modification. Hands
are a major source of contaminating RNase, so wear gloves to avoid contamination].
DEPC treatment of solutions

Add 0.1% DEPC to the solution and shake it vigorously to mix the DEPC into the solution.
Autoclave the solution for 45-60 minutes to inactivate the remaining DEPC, which may
degrade RNA during the processing. Wear gloves and use fume hood when using DEPC, as
it is suspected carcinogen. Tri Extract (Trizol) is a convenient, ready to use reagent designed
for the isolation of total RNA that is free from protein and DNA contamination. The isolated
RNA is suitable for northern blots, dot blot hybridization and in vitro translation, RNase
protection assays and poly (A+) selection.
Protocol for RNA isolation

1. Add 1 ml Tri-Extract for every 100 mg tissue and homogenize.
2. Incubate the samples for 5 min at room temperature (RT).
3. Add 200 µl chloroform per 1 ml Tri-Extract. Securely cap the tubes and vigorously shake
the tubes for 15 seconds.
4. Incubate at RT for a further 5 minutes.
5. Centrifuge the tubes at a maximum 12,000x g for 15 minutes at 4°C.
6. Following separation, a lower pink, phenol-chloroform phase, an interphase and a
colourless upper aqueous phase are visible. RNA would be exclusively in the aqueous
upper phase.
7. Transfer the aqueous phase to a fresh tube.
122
8. Add 600 µl of isopropyl alcohol for every 1 ml Tri-Extract. Incubate at RT for 10
minutes and centrifuge at a maximum 12000 x g for 10 minutes at 4°C. A gel like
translucent pellet of RNA would be precipitated on the side/ bottom of the tube.
9. Remove the supernatant and wash the RNA pellet with 70% ethanol.
10. Vortex to mix and centrifuge at 7500 x g for 5 min at 4°C. Discard the supernatant.
11. Air dry the RNA pellet for 10-15 min (don’t over dry) and dissolve in an appropriate
volume of RNase-free water. To aid the process, incubate at 55-60°C for 10 minutes.
Note: Isolated total RNA samples can be stored at 4°C for a week, but for longer period,
should be stored at 80°C.
Determination of total RNA yield and quality

The yield of total RNA obtained may be determined at 260 nm using spectrophotometer
(1A260 unit =40 µg/ml of single stranded RNA).
Purity: Pure RNA will exhibit A260/A280 ratio of 2.0. However, due to variations between
different starting materials and individual variation in performing the procedure one can
expect to obtain RNA having A260/A280 ratios ranging from 1.7-2.0. The ratio lower than this
is generally indicative of contamination.
Integrity: Integrity of purified RNA can be checked by running it on a denaturing agarose
gel.
RNA Electrophoresis (Denaturing gel)

RNA is separated using denaturing agarose gel. Guanidine thiocyanate (GTC) or 3-(N-
morpholino) propanesulfonic acid (MOPS) can be used as the denaturant. The mobility of
RNA is inversely proportional to the log molecular weights of the denatured RNA species.
Reagents
A. 10 TBE (pH 8.0)

Tris - 121.12 g
Boric acid - 58 g
0.5 M EDTA - pH (7.2) 20 ml
Adjust pH to 8.0 with HCl and final volume-with ddw to 1000 ml. Autoclave and use.
B. 1 M GTC : 118 gm in 1 litre of sterile water

C. Loading dye (2 ) - 20% formamide
40% formaldehyde
50 mM phosphate buffer
3 mM EDTA
30% 5 Ficoll blue solution
D. 5 Ficoll blue solution - 25% Ficoll 400
0.1% EDTA
0.5% SDS
E. Ethidium bromide (10 mg/ml)
123
Procedure
Weigh 0.36g of agarose and add 30 ml of 1 x TBE in a flask. Boil it to dissolve agarose and
cool to room temperature. Add 0.6 ml of 1 M GTC and 3 µl ethidium bromide (lx TBE is
prepared with DEPC treated water). Gel solution is poured into the casting tray with comb in
place. Mix RNA samples with sample buffer and heat at 65°C for 10 min. Remove the comb
carefully and place the gel tray in the electrophoresis apparatus. Pour 1 x TBE just enough to
cover the gel and load the samples in the well run the gel at 5V/cm till the dye reaches three
fourth of the gel. View under UV transilluminator and photograph. When resolved by
electrophoresis the 28S, 18S eukaryotic RNA should exhibit a near 2:1 ratio on ethidium
bromide staining indicating that no significant degradation of RNA has occurred.
Total RNA separated on agarose gel electrophoresis
124
Complementary DNA (cDNA) Synthesis
Complementary strand DNA (cDNA) synthesis followed by cloning of the expressed
sequence of gene is one of the most challenging tools in the field of molecular biology. A
number of different approaches of conversion of synthesis of double stranded copies of m-
RNA have been reported. But essentially these methods involve the synthesis of first cDNA
strand with reverse transcriptase (RNA-dependent DNA polymerase) using oligo (dT) or
random hexamer as primer, removal of RNA strand by alkaline degradation, synthesis of
second strand with Eescherichia (E-coli) DNA polymerase I or reverse transcriptase (using a
hairpin loop at the 3'end of the first DNA stand as primer) and finally removal of the loop
connecting first and second strand with single stranded specific nuclease S1. The double
stranded cDNA thus synthesized is further used for analysis.
First strand cDNA synthesis for PCR amplification
First strand cDNA synthesis kit is procured from Qiagen. This provides all the enzymes and
buffer for cDNA synthesis.
First strand cDNA synthesis
1. Total RNA - 3 µl
Oligo dT primer - 1µl
DEPC water - 8 µl
[Mix gently and spin down]
2. Incubate the mix at 70°C for 5 min. Chill on ice and collect it by centrifugation.
3. Place the tube on ice and then add
5x reaction buffer - 4 µl
Ribolock Ribo nuclease inhibitor - 1 µl
10 mM dNTP mix - 2 µl
4. Mix gently. Collect by brief centrifugation.
5. Incubate at 37°C for 5 min.
6. Add Revert AidTM H minus M-MuLV - 1 µl
7. Final volume with the helper DEPC water - 20 µl
8. Incubate the mix at 42°C for 60 min.
9. Stop reaction by heating at 70°C for 10 min or chill on ice. Keep this synthesised cDNA
at 20°C .
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Expression Profiling of Gene using Reverse
Transcription-PCR
In two-step RT-PCR, qualitative reverse transcription and PCR are performed sequentially in
two separate reaction tube. Use of oligo-dT primers is strongly recommended for the RT in
long range two step RT-PCR. First carry out a RT-reaction to generate cDNA (upto 12.5 kb
in length) followed by its amplification.
This protocol constitutes the second step of two-step RT-PCR and allows amplification of
cDNA targets upto approximately 10 kb fragments.
Important points before starting

1. Set up all reactions on ice.
2. Always use an elongation temperature at 68°C.
3. Always use denaturation conditions of 93°C for 15s. Do not exceed this temperature.
4. Use a final dNTP concentration of 500 mM.
Procedure
1. Thaw 10x long range PCR buffer, dNTP mix, primer solutions and RNase-free water.
Mix the solutions thoroughly before use.
2. Prepare a reaction mix as follows:
Component Volume in each reaction Final concentration

10x Long range PCR buffer 5 µl 1 x 2.5 mM Mg2+
2+
with Mg
dNTP mix (10 mm each) 2.5 µl 500 [mM of each dNTP]
Primer A (Forword) Variable 0.4 µ M
Primer B (Reverse) Variable 0.4 µ M
RNase free water Variable
Long range PCR mix 0.4 µl 2 units per 50 µl reaction
Template cDNA (Final Variable (50-500 ng) 50 µl
volume)
3. Mix the reaction mix thoroughly and dispense appropriate volumes into PCR tube.
4. Add 50-500 ng of cDNA template to each tube containing reaction mix.
The volume of cDNA added as template from the RT reaction should not exceed 10%
of final PCR volume.
Cycling protocol for long range PCR (0.1-10 kb)
Initial activation Q 3 min 93°C initial denaturation of template cDNA

3 step cycling
Denaturation 15s 93°C do not exceed this temperature
Annealing 30s 62oC approximately
Extension 1 min/kb 5°C below Tm of primers
(68°C, Use an extension time of 1 min/kb of cDNA)
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Number of cycles 35 Amplification of 35 cycles will produce satisfactory
results in most cases. However, the optimum number of
cycles depends on the amount of template DNA
End of PCR cycling 4°C hold
For a simplified hot start, place the tubes immediately into a thermal cycler that is preheated
to 93°C and start the cycling as mentioned above. After amplification samples can be stored
overnight at 2-8°C or for longer periods at -20°C.
Analysis of PCR products
After amplification the products are separated on 1.5% agarose gel (Agarose gel percentage
is changed depending upon the expected size of the amplicon).
Agarose gel electrophoresis
1. Weigh required quantity of agarose for 50 ml.

2. Add 50 ml of 1 x TBE and dissolve agarose by boiling.
3. Cool the solution to 50°C and add 0.5 µl of ethiduim bromide (10 mg/m1) and mix by
swirling.
4. Pour the mix to the gel mould with comb in place and allow to solidify.
5. Remove the comb carefully and immerse the gel in buffer tank.
6. Mix the PCR reaction mix with 6x loading dye and carefully load into the wells along
with a standard molecular size marker.
7. Run the gel at 80V till bromophenol dye has reached three fourth of the gel and view
under UV/Q photograph it.
127
Quantitative Expression Profiling of Gene using RT-qPCR
Introduction
A real-time polymerase chain reaction is a molecular biology technique, based on the
polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule
during the PCR, i.e. in real-time, and not at its end, as in conventional PCR. Real-time PCR
can be used quantitatively (Quantitative real-time PCR), semi-quantitatively, i.e. above/below
a certain amount of DNA molecules (Semi quantitative real-time PCR) or qualitatively
(Qualitative real-time PCR).Two common methods for the detection of PCR products in real-
time PCR are: (1) non-specific fluorescent dyes that intercalate with any double-stranded
DNA, and (2) sequence-specific DNA probes consisting of oligonucleotides that are labelled
with a fluorescent reporter which permits detection only after hybridization of the probe with
its complementary sequence.The Minimum Information for Publication of Quantitative Real-
Time PCR Experiments (MIQE) guidelines propose that the abbreviation qPCR be used for
quantitative real-time PCR and that RT-qPCR be used for reverse transcription–qPCR. The
acronym "RT-PCR" commonly denotes reverse transcription polymerase chain reaction and
not real-time PCR.
In conventional PCR, the amplified product, or amplicon, is detected by an end-point

analysis, by running DNA on an agarose gel after the reaction has finished. On the contrary,
real-time PCR allows the accumulation of amplified product to be detected and measured as
the reaction progresses, that is, in “real time”. Real-time detection of amplicon is made
possible by including in the reaction a fluorescent molecule that reports an increase in the
amount of DNA with a proportional increase in fluorescent signal. DNA-binding dyes and
fluorescently labelled sequence specific primers or probes are basically used for the
quantification process. Specialized thermal cyclers equipped with fluorescence detection
optical modules are used to monitor the fluorescence as amplification occurs. The measured
fluorescence reflects the amount of amplified product in each cycle.
Real-time PCR results can either be qualitative (presence or absence of a sequence) or
quantitative (number of copies of DNA). Qualitative results are used for the validation
purpose of gene. Real-time PCR data can be evaluated without gel electrophoresis, resulting
in reduced experiment time and increased throughput. Opportunities for contamination are
also reduced, as reactions are run and data are evaluated in a closed-tube system.
Principle
Real-time PCR is carried out in a thermal cycler with the capacity to illuminate each sample
with a beam of light of at least one specified wavelength and detect the fluorescence emitted
by the excited fluorophore. The thermal cycler is also able to rapidly heat and chill samples,
thereby taking advantage of the physicochemical properties of the nucleic acids and DNA
polymerase.The PCR process generally consists of a series of temperature changes that are
repeated 25 – 50 times. These cycles normally consist of three stages: the first, at around 95
°C, allows the separation of the nucleic acid’s double chain; the second, at a temperature of
around 50-60 °C, allows the binding of the primers with the DNA template; the third, at
between 68 - 72 °C, facilitates the polymerization carried out by the DNA polymerase. Due
to the small size of the fragments the last step is usually omitted in this type of PCR as the
enzyme is able to increase their number during the change between the alignment stage and
the denaturing stage. In addition, in four steps PCR the fluorescence is measured during short
temperature phase lasting only a few seconds in each cycle, with a temperature of, for
128
example, 80 °C, in order to reduce the signal caused by the presence of primer dimers when a
non-specific dye is used. The temperatures and the timings used for each cycle depend on a
wide variety of parameters, such as: the enzyme used to synthesize the DNA, the
concentration of divalent ions and deoxyribonucleotides (dNTPs) in the reaction and the
bonding temperature of the primers.
The amplification plot shows two phases, an exponential phase followed by a non-
exponential plateau phase. During the exponential phase, the amount of amplicon
approximately doubles in each cycle. As the reaction proceeds, however, reaction
components are consumed, and ultimately one or more of the components becomes limiting.
At this point, the reaction slows and enters the plateau phase (cycles 28–40). Initially,
fluorescence remains at background levels, and increases in fluorescence are not detectable
(cycles 1–18), even though product accumulates exponentially. Eventually, enough amplified
product accumulates to yield a detectable fluorescent signal. The cycle number at which this
occurs is called the threshold cycle, Q (CT). Since the CT value is measured in the exponential
phase when reagents are not limited, real-time qPCR can be used to reliably and accurately
calculate the initial amount of template present in the reaction. The (CT) of a reaction is
determined mainly by the amount of template present at the start of the amplification
reaction. If a large amount of template is present at the start of the reaction, relatively few
amplification cycles will be required to accumulate enough products to give a fluorescent
signal above background. Thus, the reaction will have a low, or early, (CT). In contrast, if a
small amount of template is present at the start of the reaction, more amplification cycles will
be required for the fluorescent signal to rise above background. Thus, the reaction will have a
high, or late, CT. This relationship forms the basis for the quantitative aspect of real-time
PCR. Multiplexing is the amplification of more than one target in a single reaction tube.
Currently, it is possible to amplify and quantify as many as five targets in a single tube,
depending on the features of the real-time PCR instrument. Multiplexing confers the
following advantages over single plex reactions:
Reduction in the amount of starting template required, which is important when the
amount of starting material is limited.
Reduction in false negatives, if a control target is amplified within each sample.
Increased laboratory throughput with a concomitant reduction in reagent costs.
Minimization of sample handling and associated opportunities for laboratory
contamination.
If none of these considerations are important for your assays, then single-plex reactions are
sufficient.
DNA-Binding Dyes (SYBER Green I) :

The most commonly used DNA-binding dye for real-time PCR is SYBR Green I, which
binds non-specifically to double-stranded DNA (dsDNA). SYBR Green I exhibits little
fluorescence when it is free in solution, but its fluorescence increases up to 1,000-fold when
it binds dsDNA. Therefore, the overall fluorescent signal from a reaction is proportional to
the amount of dsDNA present, and will increase as the target is amplified.
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Fluorescent Primer- and Probe-Based Chemistries
TaqMan Assays
TaqMan assays employ a sequence-specific, fluorescently labelled oligonucleotide probe
called the TaqMan probe, in addition to the sequence-specific primers. Also known as the 5'-
nuclease assay, the TaqMan assay exploits the 5'-exonuclease activity of certain thermostable
polymerases, such as Taqor Tth.
Molecular Beacons
In addition to two sequence-specific primers, molecular beacon assays employ a sequence-
specific, fluorescently labelled oligonucleotide probe called a molecular beacon, which is a
dye-labelled oligonucleotide (25–40 nt) that forms a hairpin structure with a stem and a loop
primer design.
Primer Designing
Design primers for the amplicon size of 75–200 bp as shorter amplicons are amplified with
higher efficiency. An amplicon should be at least 75 bp to easily distinguish it from any
primer-dimers that might amplify during the reaction and also avoid secondary structures, if
possible. Use programs such as mfold (http://www.bioinfo.rpi.edu/applications/mfold/) to
130
predict whether an amplicon will form any secondary structure at annealing temperature.
Avoid templates with long (>4) repeats of single bases.
For primer designing follow these parameters:

Design primers with a GC content of 50–60%.
Maintain a melting temperature (Tm) between 50ºC and 65ºC.
Avoid secondary structure; adjust primer locations outside of the target sequence
secondary structure if required.
Avoid repeats of Gs or Cs longer than three bases.
Place Gs and Cs at the terminals of primers.
Check sequence of forward and reverse primers to ensure no 3' complementarity to avoid
primer-dimer formation.
Verify specificity using tools such as the Basic Local Alignment Search Tool (BLAST)
Melting Curve Analysis

Melt-curve analysis can be used to identify different reaction products, including non-specific
products. After completion of the amplification reaction, a melt curve is generated by
increasing the temperature in small increments and monitoring the fluorescent signal at each
step. As the dsDNA in the reaction denatures (i.e., as the DNA “melts”), the fluorescence
decreases. The negative first derivative of the change in fluorescence is plotted as a function
of temperature. A characteristic peak at the amplicon’s melting temperature (Tm, the
temperature at which 50% of the base pairs of a DNA duplex are separated) distinguishes it
from other products such as primer-dimers, which melt at different temperatures.
Real time PCR analysis can be carried out using QuantiTect SYBR Green PCR kit from
Qiagen according to manufacturer's protocol. Thaw 2x QuantiTect SYBR green PCR Master
Mix, cDNA primers and RNase free water.
Requirement
Gene specific primer- Vector NTI, OligoAnalyzer, Prime3 Out Q

SYBR Green Kit (Universal) - Saf lab, Bio Rad, Qiagen
Total RNA isolation kit- Qiagen, Nucleopore, Bio Rad
cDNA synthesis kit- Fermentas, Qiagen
Qubit for quantification of nucleic acid- Invitrogen, Thermo Fischer Scientific
Real Time PCR instrument- BioRad, ABI system
Semi-skirted 96 well PCR plate- Bio Rad, Roche, Invitrogen
Sealer- Bio Rad, Thermo Fischer Scientific
Roller- Genetix, Bio Rad, Thermo Fischer Scientific
Procedure
Step-1 Total RNA Isolation- Trizol Method/ Kit
Step-2 cDNA Synthesis- Single stranded cDNA synthesis kit
Step-3 Quantification of cDNA- Nanodrop/Picodrop
Step-4 Preparation of reaction mix- SYBR Green-I kit
Step-4 Quantification of fluorescence- Real Time PCR Instrument
Step-5 Data Analysis-Pfaffl method
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Reaction mix for qPCR
Component Volume Final conc.

2 SYBER Green, Master mix 10 µl l
Forward primer (10 µM) 0.4 µl 0.2 µM
Reverse primer (10 µM) 0.4 µl 0.2 µM
cDNA (20 ng) 1 µl 1 ng
RNase free water FV to 20 µl
Real time cycle conditions
Step Time Temperature

Step-1 3 minutes 95°C
cycle 39 times Step 2 to 5
Step-2 10 seconds 95°C
Step-3 30 seconds 60°C (Based on Tm)
Step-4 5 seconds 72°C
Step-5 Plate read
52° to 95°C with an increment of 5°C per
Step-6
second followed by plate read
End
SYBR Green fluorescence chart Melting curve at the end RTqPCR
Analysis of qPCR Products

Standard curve:
The standard curve is generated by using dilution series of known amount of nucleic acid
sample or the target genes. The Ct value is inversely proportional to the log of the initial
copy number. Therefore, a standard Q curve is generated by plotting the Ct values, against
the logarithm of the initial copy numbers. The copy numbers of experimental DNA/RNA
sample can be calculated after real-time amplification from the linear regression of that
standard curve.
132
Log Starting Quantity, copy number
E=-83.O% R is 2=0. 956 slope=1. 299 y-i nt=12.
PCR Standard curve
Quantification
The Q shows a typical reading from a single PCR cycle in a real time PCR machine. The
vertical axis represents copy number (arbitrary units) and the horizontal axis shows the PCR
cycle number. The dotted threshold line is an arbitrary value, usually about 0.1 and is the
"copy number" used to determine Ct (Threshold cycle). The lower a Ct value, the more copies
are present in the specific sample. When plotted on a linear scale, the graph produces a
sigmoid curve with an exponential phase and a plateau phase. The plateau phase is really
determined by the amount of primer in the master mix rather than the nucleotide template.
Usually the vertical scale is plotted in a logarithmic fashion, allowing the intersection of the
plot with the threshold to be linear and more easily visualized. Theoretically, the amount of
DNA doubles every cycle during the exponential phase, but this can be affected by the
efficiency of the primers used. A negative control is always performed with no template to
show a lack of intrinsic fluorescence. When real time PCR is combined with reverse
transcriptase PCR (RT-PCR), mRNA can be quantified for an assessment of relative gene
expressions between tissues or genes. The amount of DNA/RNA is determined by comparing
the results to a standard curve produced by serial dilutions of a known amount of DNA/RNA.
Threshold Cycle
Fluorescence values are recorded during every cycle and represent the amount of product
amplified to that point in the amplification reaction. The more templates present at the
beginning of the reaction, the fewer number of cycles it takes to reach a point in which the
fluorescent signal is first recorded as statistically significant above background. This point is
defined as the Ct, and will always occur during the exponential phase of amplification.
Data Analysis Q (Pfaffl method)

The Pfaffl formula (Ratio = 2–ΔΔCt) was used to calculate the relative expression of genes,
where ΔΔCt = (ΔCt sample-ΔCt control); ΔCt sample = (ΔCt target-ΔCt reference) Graphs
would be plotted against control and sample.
References
Pfaffl MW (2001) A new mathematical model for relative quantification in real time RT-
PCR. Nucleic Acid Res.1: 29-45
133
Southern Blot Analysis for the Identification
of Gene Copy Number
Archana Sachdev and Monica Jolly

110012
Introduction
Southern hybridization is a technique for transfer of DNA molecules from an electrophoresis
gel to a nitrocellulose or nylon membrane, and is carried to detect specific DNA sequences
by probe hybridization. It is used to determine the copy number of transgene integration.
First devised by E. M. Southern (1975), Southern blotting results in transfer of DNA
molecules usually restriction fragments from an electrophoresis gel to a nitrocellulose or
nylon membrane so that the DNA banding pattern present in the gel is reproduced on the
membrane. The advent of Southern transfer and the associated hybridization techniques made
it possible for the first time to obtain information about the physical organization of single
and multicopy sequences in complex genomes. Southern transfer is used for homology-based
cloning on the basis of amino acid sequence of the protein product of the target
gene. Oligonucleotides are designed that are similar to the target sequence. The
oligonucleotides are chemically synthesised, radiolabeled, and used to screen a DNA library,
or other collections of cloned DNA fragments. Sequences that hybridize with the
hybridization probe are further analysed, to obtain the full length sequence of the targeted
gene. The DNA attached to the filter is then hybridized to P32 / biotin-labelled DNA or RNA
and autoradiography is used to locate the position of any bands complementary to the labelled
probe.
There is also a sensitive color Southern blot protocol biotin-labelled DNA or RNA that
allows detection of nanogram quantities of both host and pathogen DNA simultaneously in
less than two days. Host and virus genome probes were labeled with biotin-modified
nucleotides and detected using IRDye-conjugated Ab and streptavidin, eliminating the need
for radioactive label. The signals from the IRDye-conjugated Ab and streptavidin were
extremely stable and did not decay, allowing for reanalysis weeks after initial development as
well as extended storage of probes.
Solutions
1. 1% HCl
2. Denaturing solution
0.5 M NaOH
1.5 M NaCl
3. Neutralizing solution
1.0 M Tris-HC1 (pH 7.2)
1.5 M NaC1
4. 20x SSC
0.3 M Sodium citrate
3.0 M Sodium chloride (pH 7.2)
134
(a) Restriction of DNA and agarose gel electrophoresis
DNA - 5 µg
10x Buffer - 5.0 µl
Restriction enzyme - 2.5 µl (is 5U/µg DNA)
Sterile water to - 50 µl
Mix the above components in a 1.5 ml Eppendorf tube
1. Incubate the reaction mix at 37°C for 5 hr or overnight.
2. Add 5 µl of 10x dye to the sample centrifuge for 5 sec in an Eppendorf and load on to a
0.8% agarose gel prepared in 1x TBE.
3. Load λ DNA cut with HindIII and EcoRI to an adjacent well as molecular size marker.
Run the gel at 5 V/cm till the bromophenol dye has reached 3/4th of the gel.
4. Visualize the DNA bands under UV transilluminator and place a ruler next to the gel to
be able to determine the fragment size later on.
Restriction digestion of genomic DNA visualized on agarose gel

electrophoresis
(b) Transfer of DNA to nylon membrane

After visualizing the gel under UV it is kept in a glass tray and is shaken slowly at room
temperature on a shaking platform in the following way:
15 min with 1% HCl
15 min with denaturing solution
15 min with neutralizing solution
15 min in 20x SSC.
While the gel is being washed in the last solution, start preparing the blotting assembly
and positively charged nylon membrane.
Set up the assembly on a levelling table and check that the surface is levelled. Add 20x SSC
to a glass reservoir to the required depth and keep a glass plate over it. Cut a piece of
Whatman 3 MM filter paper and wrap the glass plate with it. Keep it in the dish with the
135
ends of the paper dipping in 20x SSC (serves as a wick). Wet the paper with 20x SSC and
remove all air bubbles entrapped by rolling a glass rod over it. Cut a piece of nylon
membrane 2-3 mm larger than the gel and float it on sterile water for 10 min followed by 20x
SSC for 10 min. (when using a different membrane, follow the company suggested protocol
for transfer. For example: with Nytran 10X, SSC is recommended for the transfer buffer,
while 20X SSC is used for nitrocellulose)
Place the gel slowly over the filter paper in such a way that the wells are now facing the glass
plate. The nitrocellulose paper can be marked with a pencil. Remove air bubbles by rolling a
glass rod over the gel. Take the nylon membrane using a pair of forceps and place it over the
gel slowly and remove air bubble. Place a sheet of 3 MM paper wet in 20x SSC over the
membrane and also 3 dry sheets of Whatman 3 MM paper. Keep stack of absorbent paper
towels to about a height of 6 cm over it. Finally keep a glass plate over it and weigh it down
with 500 g weight. To prevent short circuiting of fluid between the paper towels and 3 MM
paper under the gel, surround the gel with a water tight border of Saran Wrap.
Allow transfer of DNA to proceed for 6 hrs to overnight. Small fragments of DNA (<1
kb) transfer from 0.8% gel within an hour or two, while transfer of DNA >15 kb takes 15 hrs or
more. After the blotting is complete remove the stack of absorbent papers, filter papers and
carefully mark the wells of the gel with a soft pencil. Wash off any adhered agarose from the
membrane by washing it in 5x SSC. Air-dry the membrane and bake at 80°C for 1-2 hrs in an
oven. It can now be stored indefinitely at room temperature protected inside a filter paper.
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(c) Preparation of probe by oligo-labelling (using Decalabeling kit, MBI Fermentas)
This method is based on the priming of the polymerase reaction on the template DNA with
random hexa nucleotide primers. The complementary strand is synthesized from the 3' end of
the primer with the help of the large fragment of DNA polymerase I exonuclease minus
(Klenow Fragment exo-) in the presence of labelled deoxy ribonucleoside triphosphate.
DNA template (100 ng) - 10 µl
Decanucleotide in 5x reaction buffer - 10 µl
Nuclease free water - 20 µl
Mix the above components in a microfuge tube and vortex for a short duration and spin down the
contents in a micro-centrifuge for 3-5 sec.
1. Incubate the tube in a boiling water bath for 5-10 min and cool it on ice. Spin down
quickly.
2. Based on the availability of labelled triphosphate (dATP or dCTP) use Mix A or C
respectively.
3. Add the following components in the tube:
Mix A or C 3 µl
32 32
α-P dATP or α-P dCTP 6 µl (10 µ Ci/ml)
-
Klenow fragment exo 1 µl (5 U/µl)
Mix the contents and spin down in a microfuge. Incubate for 10 min at 37°C.
4. Add 4 µl of dNTP and incubate for 5 min at 37°C.

5. Stop the reaction by adding 1 µl of 0.5 M EDTA (pH 8.0).
6. The labelled DNA is used directly for hybridization after denaturing by heating in a
boiling water bath for 10 min.
(d) Hybridization
Solutions
1. Pre-hybridization solution
0.5 M Na2HPO4 20 ml
20 % SDS 14 ml
Sterile water 6 ml
2. 20x SSC
3. 20 % SDS
Procedure
1. Keep the nylon membrane in a hybridization bottle and pour pre-hybridization solution 5
m1/100 cm2.
2. Keep the bottle in a hybridization oven set at 65°C and incubate for 1-2 hr with slow
rotation (see that the tubes are kept balancing in the roller).
3. Pour off the solution from the bottle and add fresh pre hybridization solution (2 ml/100
cm2) to which denatured probe has been added.
4. Continue incubation at 65°C with slow rotation in the hybridization oven overnight.
137
(e) Washing the membrane and autoradiography
i. After completion of hybridization, remove the solution and add 2 x SSC and 0.1% SDS
(250 m1/100 cm2) to the bottle.
ii. Keep it on the shaker and shake vigorously for 5 min at room temperature.
iii. Discard the wash solution and repeat it twice.
iv. Continue washing of the filter two times with 0.2 SSC + 0.1% SDS at 50°C. If more
stringency is required washing may be repeated with 0.1 SSC + 0.1 % SDS at 65°C.
v. Remove the membrane from the solution. Air-dry it and expose to X-ray film at -70°C.
vi. Developing the film after appropriate number of day’s exposure using X-ray film
developer and fixer.
Note:
1. The Tm of the hybrid formed between the probe and its target may be estimated using the equation:
Tm = 81.5°C - 16.6 [(log10, (Na+)] 0.41 (% G + C) - 0.63 (% formamide) - (600/1) where 1 = the
length of the hybrid in base pairs.
This equation is valid for:
a. Concentrations of Na+ in the range of 0.01 M to 0.4 M. It predicts Tm less accurately in
solutions of higher (Na+).
b. DNAs whose G + C content are in the range of 30% to 75%.
2. The Tm of a double stranded DNA decreases by 1-1.5°C with every 1% decrease in homology.
3. The depression of Tm in solutions containing formamide is greater for poly (dA :dT) (0.75°C/1%
formamide) and less for DNAs rich in poly (dG : dC) (0.05°C/1% formamide).
4. To determine the time of half renaturation (Cot1/2) for any probe following formula can be utilized:
where,
x = weight of the probe added (in µg)
y = its complexity (for most probes, complexity is proportional to the length of the probe in kb)
z = the volume of reactions (hybridization solution) (in ml)
Normally the hybridization is allowed to proceed for a time sufficient to enable the probe in solution to
achieve 1-3 x Cot1/2. After hybridization for three times Cot1/2 has been reached, the amount of the probe
available for additional hybridization to the filter is negligible.
138
Flow Chart of Southern Hybridization
Restriction of DNA and agarose Transfer of DNA to Nylon Preparation of probe by oligo- Hybridization , Washing of
gel electrophoresis Membrane labelling Membrane and Autoradiography
•Incubate the reaction • cut off the well and cut the •Mix the following •Keep the nylon membrane
upper left corner (for components in a microfuge in a hybridization bottle
mix at 37°C for 5 hr or orientation).
overnight. tube(DNA template, •Add pre-hybridization
• Place for nicking [5 ml of 10 M Decanucleotide, Nuclease
HCl in 200 ml of dH2O] 10 solution
•Add 5 µl of 10x dye to free water)
minutes rocking. •Keep the bottle in a
the sample centrifuge •Vortex shortly and spin hybridization oven
• Remove Nicking solution and
for 5 sec add enough Neutralize Buffer to down the contents in a •Pour off the solution and
•Load on to a 0.8% cover the gel and rock solution micro-centrifuge for 3-5 sec add fresh pre hybridization
agarose gel prepared in at speed 10 for 30 minutes. •Incubate in a boiling water solution
• Take (9 x 14 cm) nylon bath for 5-10 min and cool
1x TBE. membranes, wet by dipping in •Incubate at 65°C with slow
it on ice. rotation in the
•Load λ DNA cut with dH2O for 10 minutes followed
Hind III and EcoRI by 10 minutes in Neutralize •Based on the availability of hybridization oven over-
(NB) buffer. labelled triphosphate night
•Run the gel at 5 V/cm • Set up Transfer (dATP or dCTP) •After completion of
till the bromophenol • Invert the gel casting unit upon •Use Mix A or C respectively. hybridization, remove the
dye has reached 3/4th of the gel box. •Add the following solution and add 2x SSC
the gel. • Cut out a strip of filter paper (at components in the tube: and 0.1% SDS (250ml/100
least 9 cm wide) and drape over
•Visualize the DNA bands the gel casting. •(Mix A or C , αP32dATP or cm2)
under UV trans- • Wet the filter paper with αP32dCTP, Klenow fragment •Shake vigorously for 5 min
illuminator Neutralize buffer. exo) at room temperature
•take the gel picture • Fill up with NB buffer as well till •Mix the contents and spin •Discard the wash solution
the wells on the gel box. down in a microfuge. and repeat it twice
• Place the gel face down on the Incubate for 10 min at •Continue washing of the
filter paper. 37°C. filter two times with 0.2x
• Place membrane on gel.
•Add 4 µl of dNTP and SSC + 0.1% SDS at 50°C
• Wet first filter paper and add to incubate for 5 min at 37°C
the 5-6 inches stack of filter •Remove the membrane
paper sized to gel (about 9 x 14 •Stop the reaction by adding from the solution.
cm) 1 µl of 0.5 M EDTA (pH 8.0) •Air-dry it and expose to X-
• Place a glass plate and put •The labeled DNA is used ray film at -70°C.
weight 500gm on stack. Leave directly for hybridization •Developing the film after
overnight. after denaturing appropriate number of
day’s exposure
(d) Preparation of probe by biotin-labelling (Biotin 3´ End DNA Labeling Kit,Thermo

scientific)
Biotin
Labeling Probe DNA by non radioactive / biotin:

In this experiment, the DNA to be used as a probe in the Southern hybridization is labeled
with biotin. Through an enzymatic reaction, nucleotides to which biotin has been attached
will be incorporated into the probe DNA (Fuccillo, 1985). Double stranded DNA to be
labeled is treated with DNaseI and E. coliDNA polymerase I. DNaseI makes random nicks
(breaks in the phospho-diester backbone) in DNA. E. coliDNA polymerase I recognizes such
nicks and removes nucleotides one at a time from the 5′side of the nick and adds nucleotides
to the 3′end of the nick (resulting in movement—translation—of the nick). Also provided in
this reaction are the precursors DNA polymerase incorporates into DNA, the deoxy
nucleotide triphosphates-dNTPs. In our reaction, dCTP, dGTP, dTTP, and biotin-7- dATP (a
dATP analog with biotin attached to the 6-position of the purine base by a 7-atom linker) are
139
provided. By the action of DNA polymerase I, the nucleotides in the DNA are removed and
(replaced by new ones), including biotin-7-dATP. A biotin-labeled DNA probe is made in
this way
Detection of Biotin-labeled Probes: follow the instruction as per the manufacturer’s protocol
mentioned in the kit Biotin Chromogenic Detection kit (Thermo Scientific, USA).
To summarize the steps:
The membrane was hybridized with biotin labelled

probe in hybridization solution
The biotin-labeled single stranded (ss) DNA probe

hybridized to blot
streptavidin-alkaline phosphatase (SA-AP)

conjugate
Detection of hybridized probe: Blocking BSA and

Add SA-AP
Add chromogenic substrate to (SA-AP) for colour
Procedures
Membrane Blocking
In the plastic box, wash (or rehydrate if dried) the hybridized membrane for 1 minute
in buffer. Use enough volume to cover membrane completely.
Incubate membrane for 1 hour at 65°C in Buffer 2 (pre-warmed to 65°C) in the sealed
box.
(Note: Buffer 2 contains the protein BSA. Soaking the membrane in BSA saturates non-
specific protein binding sites on the membrane. This step minimizes the non-specific binding
of SA-AP to the membrane.)
The membrane may be dried at this point (air dried or dried in a vacuum oven 80°C
10–20 min) and stored for months, or continue with the procedure. Binding of
Streptavidin-alkaline Phosphatase Conjugate.
Again, use a plastic box. If membrane is dry, soak membrane in Buffer 2–10 min to
rehydrate completely.
In a polypropylene tube, immediately before use, add 7 μl SA-AP conjugate(1 mg/ml)
to 7.0 μl of Buffer 1. Incubate membrane in diluted SA-AP conjugate for 10 minutes.
This time occasionally pipet the solution over the membrane. Pour off the solution.
Wash membrane with 250 ml Buffer 1. Gently rock for 15 minutes. Pour off Buffer 1.
Wash membrane with an additional 250 ml of Buffer 1.
Wash membrane in 250 ml of Buffer 3 for 10 minutes.
140
(Note: Buffer 3, pH 9.5, provides the appropriate conditions for alkaline phosphatase enzyme
activity).
Addition of Chromogenic Substrate to Visualize DNA(prepare dyes immediately
before their use)
In a polypropylene tube, add 33 μl NBT solution to7.5 ml of Buffer 3. Mix gently by
inverting the tube. Add 25 μl BCIP solution, mix gently. (The use of a polypropylene
tube rather than a polystyrene tube is recommended because biotin has a tendency to
stick to polystyrene, but less so to polypropylene.)
Caution: The dye solutions contain dimethylformamide, which is harmful if inhaled,
swallowed, or absorbed through the skin. Wear gloves and wash hands thoroughly after use.
Incubate the membrane in dye solution in the small plastic box, with the lid on. Let
the color development proceed in the dark or in very low light. Check for color
development every 5–10 min. DNA bands should be visible within 10 minutes to 3
hours. (Longer incubations may result in increased background.) The bands will be
clearer on one side of the membrane than the other.
To stop color development reaction, wash membrane in 20 mM Tris pH 7.5/0.5 mM
Na2-EDTA. Let membrane air dry. Store membranes away from strong light to
prevent color fading. To some extent, wetting the membrane in water helps bring out
the color again. These membranes cannot be reprobed because NBT and BCIP
cannotbe removed from the nitrocellulose. However, it is possible to reprobe with a
different sequence and detect additional bands.
Problems and Remedies Transfer/Hybridization: Poor Transfer
S. No. Problem Troubleshoot

1 Bubbles and spots of no transfer Treat filter carefully - do not touch with bare
hands, roll out bubbles while setting up blot.
Gloves should be worn when
handling the nitrocellulose pa
per.
2 Bottom filter weaker (and Avoid excess weight in blotting; use bottom
Blurrier) than top filters (which may be weaker due to less DNA
transferred) with high-copy number of probes;
hybrids with total mtDNA or cpDNA to assess
bad spots.
3 Double images Don't slide gel or filters around while setting up
the blot.
4 Large fragments weak and poorly Use acid depurination in transfer protocol.
transferred.
5 Non-specific background Don't let filters dry in wrapping, washing and
exposing, improper pre-hybridization
(especially if large, bubble-shaped blotches),
strip and repeat, use larger volume of pre-
hybridization solution.
141
6 Hybridization to contaminating Use isolated inserts; avoid vector
vector DNA contamination of DNAs.
7 Inability to strip completely Let filters decay, use low-copy number and
previously hybridized probe. more divergent probes first, high-copy number
and conserved probes last; don't let filters dry
after probing.
References
Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. and
Struhi, K. (1987) Curr. Protocols Biochem. John Wiley & Sons (5 November 1975).
Towbin; Staehelin, T; Gordon, J; et al. (1979). "Electrophoretic transfer of proteins from
polyacrylamide gels to nitrocellulose sheets: procedureandsomeapplications". PNAS.
76 (9):4350/4. doi:10.1073/pnas.76.9.4350. PMC 411572 . PMID 388439.
Reed, K.C. and Mann, D.A. (1985) Rapid transfer of DNA from agarose gels to nylon
membranes. Nucl. Acids Res. 13: 7207-7221.
Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. and
Struhi, K. (1987) Curr. Protocols Biochem. John Wiley & Sons
Sambrook J,Fritsch EF and Maniatis T (1989) Molecular Cloning. A Laboratory Manual.
Cold Spring Harbor,NY: Cold Spring Harbor. Fucillo, D. A. (1985) Biotechniques 3, 494-501
Eweida M1, Sit TL, Sira S, AbouHaidar MG.( 1989) Highly sensitive and specific non-
radioactive biotinylated probes for dot-blot, Southern and colony hybridizations. J Virol
Methods. 26(1):35-43.
Mackey, J., Darfler, M.; Nisson, P.; and Rashtchian, A. (1993) Use of random primer
extension for concurrent amplification and non-radioactive labeling of nucleic acids. Anal.
Biochem. 212: 428-435.
142
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Biochemistry: A Practical Manual

Book · March 2019

Suresh Kumar Shelly Praveen

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Nutrigenomics View project

The user has requested enhancement of the downloaded file.

Suresh Kumar, Shelly Praveen and Aruna Tyagi

Division of Biochemistry, ICAR-Indian Agricultural Research Institute, New Delhi-

Where the symbol p denotes “negative logarithm of”

A pH Meter is an instrument that measures the hydrogen-ion concentration or pH of

Materials, Reagents and Solutions

How to calculate volume of solution to be diluted:

Vinutha T. and Veda Krishnan

The critical parameters

Absorbance corresponds to 0.1 ml of test sample = Y mg of glucose

Spectrophotometer capable of measuring absorbance in the 540 nm region.

In 1000 ml beaker dissolve 10 g of 3,4-dinitrosalicyclic acid in 200 ml H2O. Followed

 Weigh 100 mg of the sample into a boiling tube.

Amount of carbohydrate present in 100 mg of the sample= mg of glucose/Volume of test

Powdered rice grain,

Sodium maleate buffer (0.1 M, pH 6),

AACC International (2000) Approved methods of the American Association of Cereal

Vinutha T. and Navita Bansal

Hexane or diethyl ether/petroleum ether (40-60 oc).

(b) Percolation method

1. Anhydrous sodium sulphate

Kartha, A.R.S and Sethi, A.S (1957). Ind.J.Agric.Sci. 27:211.

1. Accurately weigh 1-10 g oil in a clean dry 100 ml conical flask.

Vinutha T, Navita Bansal

If oil consists of triglycerides with a high percentage of unsaturated fatty acids

1. 0.1 N sodium thiosulfate

1. Weigh 50-100 mg oil into an iodine flask and dissolve in 25 ml of carbon

Division of Biochemistry, ICAR-Indian Agricultural Research Institute, New Delhi-

1) Weigh 2 to 5 g of fresh sample according to their expected β-carotene content.

Preparation of Standard graph

Standard curve of β-carotene.

Karnjanawipagul, P., Nittayanuntawech, W., Rojsanga, P., and Suntornsuk L.

Rodriguez-Concepcion M., Stange, S. (2013) Biosynthesis of carotenoids in

Engler, M., Hammann, S., Vetter, W. (2015) Isolation of β-carotene, α-carotene

Vitamin C is a highly water-soluble natural acidic bioactive compound having strong

Red: DCP ox + 2 e− + 2 H+ → DCP red (2)

Overall: vitamin Cred + DCPox → vitamin Cox + DCP red (3)

So, vitamin C is oxidized by DCP in a 2 e–/2 H+ transfer

Extraction of ascorbic acid from sample

Estimation of ascorbic acid in sample

Division of Biochemistry, ICAR-Indian Agricultural Research Institute, New Delhi-

Chromatophore λmax (nm)

Estimation of Anthocyanins by HPLC

Figure: Chemical structure of cyanidin-3-glucoside

Materials, Reagents and Solutions

HPLC instrument conditions have to be set accordingly before analysis.

Dissolve varied concentrations of cyanidin-3-glucoside (100-1000 ppm) in 20μl acidified

Figure: Chemical structure of (A) Daidzein (B) Genistein

Materials, Reagents and Solutions

3. Sample will be extracted with 5 ml of 80% ethanol containing 1 ml HCl in a boiling

HPLC instrument conditions have to be set accordingly before analysis.

Materials, Reagents and Solutions

 Cut the plant material into small pieces

For example, if the OD value for a test

It is the intensity of this purple colour which is measured spectrophotometrically

Estimation of Free Amino acids

Ninhydrin + alpha-amino acid Hydrindantin + Decarboxylated Amino acid