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LAB MANUAL
School of Biotechnology
International University – VNU-HCMC
CONTENT
PART I. PRACTICAL EXPERIMENTS
EXPERIMENT 1:
Protein quantification applying Hartree-Lowry assay
EXPERIMENT 2:
Exploring the enzymatic activity of bromelain
EXPERIMENT 3:
Soluble carbohydrate quantification applying Anthrone assay
EXPERIMENT 4:
Quantitative determination of calcium in powdered milk
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PART I. PRACTICAL EXPERIMENTS
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EXPERIMENT 1
Protein quantification applying Hartree-Lowry assay
1. Principle
Protein quantification is highly essential in biochemical research. Several assays
have been developed; however, each has limitation due to biochemical separation
process and purposes of experimenters. Basing on differences in amino acid
content of distinct proteins, currently experimental condition, experiences of
experimenters, protein of interest and their amounts, suitable assays will be
applied to obtain the best outcome with acceptable error. Before deciding which
assay will be used, considering its sensitivity, accuracy, interfering substances and
available timing are highly recommended.
The table below shows you some widely used assays of protein quantification.
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The purified source of the protein to be quantified should be chosen as the
standard one to reduce result’s errors. In case of the proteins of interest whose
sources have not been isolated, purified or commercially sold, the other proteins
having similar structures or coming from the same family protein should be chosen
as alternatives, instead to obtain a high similar color yield.
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2. Equipment and chemicals
2.1 Equipment
+ Test tubes
+ Pipettes (1mL, 2mL, and 5mL) and pumps
+ Volumetric flasks (50mL, 100mL)
+ Beakers (50mL, 100mL)
+ Graduated cylinder (50mL)
+ Falcons (50mL)
+ Filter paper (11mm)
+ Spectrophotometer
+ Centrifuges
2.2 Chemicals
+ 0.1% Albumin solution
+ Solution A
+ Solution B
+ Solution C
+ Folin-Ciocalteu reagent
Solution A
Get 2g of Na2CO3 and dissolve it in 0.1 M NaOH to make 100mL.
Solution B
Get 0.5g of CuSO4.5H2O and dissolve it in 1% Sodium Citrate to make 100mL
Solution C
This solution can only be used within a hour, and it is the mixture of solution A and
solution B at a rate 49:1. When the solution C loses its pale-blue color, it cannot be
used any more.
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3. Practical procedures
3.1 Preparation of sample
Step 1: Get soybeans and pulverize them by blender or stone mortar.
Step 2: Take an exact amount of 5g pulverized soybeans and put it into a stone
mortar.
3.2 Extraction:
1st time:
Step 1: Get about 40mL of distilled water and put small amount of water into
the stone mortar and grind down the sample.
Step 2: Then put the rest of water to the stone mortar and grind down the
sample carefully.
Step 3: Put the extract solution into a beaker. The grounds of the soybean is
still kept in the stone mortar.
2nd time:
3rd time:
Step 4: After 3 times of extraction, you can use filter papers (takes about 1
to 1.5 hour) or centrifuge (5000rs/m for 10 minutes) to remove the
remained grounds out of the extract solution.
Step 5:
5.1: After removing remained grounds, extracted protein solution will be
poured into volumetric flask of 100mL.
5.2: Then, distilled water is added into the volumetric flask to reach the
marked level. This is your original protein solution or 100-diluted solution.
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5.3: You need to make 10,000-diluted solution by dissolving 1mL of 100-
diluted solution in 99mL of distilled water.
Step 2: The 0.1% albumin solution is diluted with the different amount of water
to make the protein solutions with different concentrations (0, 50, 100, 150,
200, and 250μg/mL).
To get the expected result, we must follow the procedure in the table below.
Tube Number 1 2 3 4 5 6
Step 2.1 0.1% Albumin solution (mL) 0 0.5 1.0 1.5 2.0 2.5
Step 2.2 Distilled water (mL) 10 9.5 9.0 8.5 8.0 7.5
Step 2.3 Shake the tube well
The concentration of each tube (μg/mL) 0 50 100 150 200 250
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After finishing making test tubes of protein solution followed the table above, you
continue to make test tubes of standard protein solutions followed the table below.
Step 3: 10 test tubes are numbered respectively from 1’ to 10’.
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4. Results and data analysis
OD
ΔOD
Protein concentration
0 50 100 150 200 250 x y
(μg/mL)
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Step 3: Click insert bar Black mark all table Choose Insert scatter
scatter
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Step 6: Choose “display equation” on chart and “display R-squared value” on
chart
4.3 How to calculation the gram of protein that contains in 100 gram of
soybean
Step 4: Use the rule of three to determine the protein concentration containing
in 10,000-diluted sample solution and 100-diluted sample solution.
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2. Equipment and chemicals
2.1 Equipment
+ Test tubes
+ Pipettes (1mL, 2mL, and 5mL) and pump
+ Beakers (500mL, 250mL and 100mL)
+ Falcons (50mL)
+ Filter paper (11mm)
+ Spectrophotometer
+ Centrifuges
2.2 Chemicals
+ 0.5M NaOH solution
+ 0.2M HCl solution
+ 5% Trichloroacetic acid solution
+ 2% Casein solution
+ Tyrosine solution
+ Folin-Ciocalteu reagent
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3. Practical procedures
3.1 Preparation of sample
Step 1: Choose fresh, moderately ripe pineapple to discard, cut into the pieces
and then pulverize it by blender or stone mortar.
Step 2: Use filter papers (takes about 1 to 1.5 hour) or centrifuge (5000rs/m
for 10 minutes) to remove the remained grounds out of the pineapple juice.
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Pineapple solution
0 0.2 0
containing bromelain
Shake the tubes well at 350C for 10 minutes
5% Trichloroacetic acid 0 5 0
Pineapple solution
0.2 0 0.2
containing bromelain
Step 2: Shake each tube well and keep them still for 10 minutes at 250C, then
filter the tubes.
Step 3: Transfer 2mL of filtered solution of each tube into new tubes
Step 4: Add 5mL of 0.5M NaOH solution and 1mL of Folin-Ciocalteu reagent
Step 5: Shake the tubes well, then put the tubes at the stand for 15 minutes and
measure the A578nm or A620nm.
Figure 2.3
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4. Results and data analysis
450. ODM 1 x1
(UI / mL)
ODT 10 x2
Where:
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EXPERIMENT 3
Soluble carbohydrate quantification applying Anthrone assay
1. Principle
In the anthrone assay, carbohydrate is dehydrated by using concentrated sulfuric
acid to form furfural, which in turn condenses with anthrone (10-keto-9,10
dihydroanthracene) to form bluish-green complex which can be measured
colorimetrically at wavelength of 620-630 nm by spectrophotometer. This a rapid
and convenient assay for determination of hexoses, aldopentose and hexuronic
acid, either in the free from or in form of polysaccharide. In this assay, the reagent
preparation does not require the addition of distilled water, and anthrone is directly
dissolved at a 2% concentration in concentrated sulfuric acid. This acid is powerful
dehydrating agent involved in dehydrating sugars leading to formation of furfural,
which condenses with anthrone to give the colored product.
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2. Equipment and chemicals
2.1 Equipment
2.2 Chemicals
+ Alcohol 90o
+ Alcohol 80o
+ Anthrone reagent
+ 0.01% Glucose solution
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3. Practical procedures
3.1 Preparation of sample
Step 1: Take 2g of pulverized raw material and put it into the beaker of 50mL.
Step 3: Put the beaker in the water bath at 80oC. Using the stirring rod to stir the
solution well during the heating process.
Step 5: Add 10mL of alcohol 80o into the beaker of grain and do the same at step
3 and 4. Alcohol can be vaporized naturally or by providing heat slight to the
beaker. Do step 5 2 times.
Step 7: Dilute the 100-diluted solution 100 times to get 10,000-diluted solution
because the amount of carbohydrate is unknown. If the carbohydrate content very
high, the solution need to be diluted more until the measured point is in range of
calibration curve.
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Tube number 1 2 3 4 5 6 7 8 9 10 11
0.01% Glucose (mL) 0 0 1 2 3 4 5
Distilled water (mL) 5 5 4 3 2 1 0
Sample (mL) 5 5
The concentration in each
0 0 0.1 0.2 0.3 0.4 0.5 x y
tube
Step 2: Put slowly 10 mL of Anthrone reagent into each tube. Let the reagent flow
along the inside-surface of the tube.
Step 3: Stir the solution very slowly by a glass stick. Then boil all tubes in hot
water for 7.5 minutes. After that, put all tubes in cool water immediately.
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Figure 3.5a Figure 3.5b
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EXPERIMENT 4
Quantitative determination of calcium in powdered milk
1. Principle
Oxalate ammonium will precipitate all calcium ion in any solution when the
experimenter set up all following conditions severely:
This method can be used for calcium quantification in milk, blood sample, urine,
food, etc. and the reactions are performed as following:
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2. Equipment and chemicals
2.1 Equipment
+ Racemic crucibles
+ Muffle furnace
+ Desiccator
+ Burette
+ Erlenmeyer flasks (250mL)
+ Beakers (100mL)
+ pH meter
+ Filter papers (11mm)
2.2 Chemicals
+ Powdered milk
+ Saturated (COONH4)2
+ Concentrated HCl
+ Methyl red
+ 0.1M NH4OH
+ Acetic acid
+ Saturated Calcium chloride
+ 1N H2SO4
+ 0.02N KMnO4
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3. Practical procedures
3.1 Preparation of sample
Step 1: Pretreat milk sample of 0.5g by burning them with absolute ethanol or
dehydrate them with concentrated sulfuric acid.
Figure 4.1
Step 4: Let the milk ash sample cool down and put them into desiccator over night
before performing experiment.
Step 1: Take three milk ash samples contained in three separated racemic
crucibles out of the desiccator and add 5mL of distilled water, and then 5 drops of
concentrated chlohydric acid.
Step 2: Mix well and transfer these solutions separately into 3 different beakers
of 250mL to adjust pH.
Step 3: Add 10-15 drops of methyl red and carry out the neutralization by 0.1
ammonium solution.
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Step 4: Adjust the pH of the solution to from 5 to 5.2 by acetic acid of weak
concentration. At that point, the solution color is orange-pink. Due to the
instrumental error, each pH meter will give a different value of pH. Therefore,
observe the color of solution is also very important. Do not over-rely on any type
of machine.
Step 5: While heating theses beakers by water bath, stirring these solutions and
fill 2-3mL of saturated (COONH4)2 solution.
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Step 6: Continue to provide heat to these beakers and then mix these solution
well for 30 seconds.
Step 8: Keep the beaker in the basin of water for about 30 minutes.
Figure 4.4
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Step 10: Use distilled water to wash the filter paper to know whether (COO)22-
ions are all eliminated or not. To check it, we use saturated calcium chloride
solution.
Figure 4.6
Using saturated calcium chloride solution to check the remained ion (COO)22
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Figure 4.7a Figure 4.7b
The color of sample solution after checking in case of remaining ion (COO) 22
Step 11: Collect precipitate retained by filter paper and put them into Erlenmeyer
flask
Step 12: Add 20mL of 1N sulfuric acid solution into each Erlenmeyer flask and
heat them in water bath with temperature of 70oC for 1 minute.
Step 13: Titrate the solution with 0.02 N potassium permanganate solution to
determine the concentration of (COO)22- ion in the solution.
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Step 14: Use the rule of three to calculate the amount of calcium ion in the
solution and in the sample.
Mn7+ + 5e → Mn2+
The relation between the concentration (M) and the concentration (N) is:
Step 2:
V×4×10-3 (mol)
Where
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V: the volume (L) of 0.02 N KMnO4 that is used to determine the quantitative
measurement
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PART II: REVIEW QUESTIONS
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EXPERIMENT 1
1. Why do we need to add Folin-Ciocalteu reagent into protein solution?
2. What is the principle that spectrophotometric method based on?
3. What is the role of Albumin in this experiment?
4. What are two reactions used for developing intensely blue colour?
5. Why do we need to make blank solution?
6. What is the disadvantage of Hartree-Lowry method?
7. Why the amount of protein of interest need to be firstly considered before choosing
quantification assay?
8. What will you do if you have to quantify protein A but you cannot buy the
commercialized standard protein A?
9. Student A says that he can insert any value of optical density in the equation of
standard curve to determine the corresponding value of protein content. Does the
student say it right or wrong? Explain why?
10. Student B says that in this practical, he can only determine relatively the total
protein content in extracted protein solution. Does the student say it right or wrong?
Explain why? Assuming that he is right, what will he do firstly before he want to
quantify mostly exactly his proteins of interest?
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EXPERIMENT 2
1. What fruit was bromelain extracted from? What is the main function of bromelain?
2. What are crucial factors which can have effects on enzymatic activity?
3. Are fixed times important for exploring/studying the enzymatic activity? Explain
why.
4. Why is trichloroacetic acid added into tube of Tyrosine and tube of water
immediately right after putting casein solution into the test tubes while with respect
to tube of sample, trichloroacetic acid is added at the end of the process?
5. Why shouldn’t we add Tyrosine into tube of sample?
6. How can the activity of enzyme be determined in this experiment?
7. Student A says that when doing this practical, he can prepare the tubes of tyrosine
and tubes of water first and then measure the optical density in order to save time.
Does he says it right or wrong? Explain why.
8. Student B says that the more he add bromelain, the faster the catalytic reaction
occurs. Does he says it right or wrong? Explain why.
9. Student C says that if he can purify and recover the bromelain used in this
practical, he can continue to use it for the catalytic reaction next time in order to save
the enzyme. Does he says it right or wrong? Explain why.
10. Student D says that there is a paradox in enzymatic activity. He supposes that
protease can degrade itself because it is also a protein. Does he says it right or wrong?
Explain why.
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EXPERIMENT 3
1. What is the solvent of Anthrone reagent?
2. When the dried samples are used, why we need to use the fewer amounts
compared to the raw ones.
3. Why alcohols are used to extract carbohydrate?
4. How long does Anthrone reagent can be kept for using?
5. Why anthrone reagent needs to be kept cold before using?
6. Why test tubes must be put in ice-water beaker/container when adding Anthrone
solution?
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EXPERIMENT 4
1. What is the role of Oxalate ammonium?
2. Why do we need to use Methyl Red?
3. Why do we use acetic acid to adjust pH instead of strong acid such Chlorohydric
acid or Sulfuric acid?
4. Why don’t prepare Potassium Permanganate solution before lab work so far?
5. Why do we need to eliminate all the (COO)22- ion out of the calcium?
6. Why does powdered milk must be pre-treated before heating in the muffle furnace?
7. Student A says that Ca2+ is an analyte and KMnO4 is a titrant. Does he says it right
or wrong? Explain why.
8. Why calcium chloride solution must be saturated?
9. Why don’t we use non-heated powdered milk to quantify calcium content?
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PART III: MAKING SOLUTION IN
BIOCHEMISTRY LAB
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EXPERIMENT 1
Folin-Ciocalteu reagent
Weigh 100g Sodium tungstate and 25g Sodium molybdate. Then, add 700mL distilled
water and 50mL concentrated orthophosphoric acid (83-85%). Stir well and add
100mL of concentrated chlohydric acid (37-27%). Stir well and reflux for 10 hours.
After refluxing, add 150g of lithium sulfate into the solution and wait until it
completely dissolved. Stir well and then add 10mL bromine solution of 3-5 drops of
pure bromine liquid. Finally, reflux for 30 minutes in the fume hood and cool the
solution at room temperature.
If the solution is not clear, filter them. The well-made solution has yellow-orange
color. If the solution turns into yellow-green, it cannot be used. Keep Folin-Ciocalteu
reagent at 4oC (in the fridge). This solution can be kept and used within 1 year.
Solution A
Get 2g of Na2CO3 and dissolve it in 0.1 M NaOH to make 100mL.
Solution B
Get 0.5g of CuSO4.5H2O and dissolve it in 1% Sodium Citrate to make 100mL
Solution C
This solution can only be used within a hour, and it is the mixture of solution A and
solution B at a rate 49:1. When the solution C loses its pale-blue color, it cannot be
used any more.
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EXPERIMENT 2
2% Casein solution
Get 2g of casein, 36g urea and 8mL of 1M NaOH to dissolve it in 40mL of distilled
water. Keep the solution at 250C for 60 min, add 10mL of 1M KH2PO4 buffer solution.
Then, adjust the pH to 6 with 2M HCl. Finally, add distilled water to the point of
100mL and keep it in the fridge.
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EXPERIMENT 3
0.01% Glucose solution
Take 0.01g of glucose that has dried in the desiccator, then dissolve in 100ml of
water.
Anthrone reagent
Dissolve 2g of anthrone in 1 L of concentrated sulfuric acid. Then, keep anthrone
solution in the fridge. This reagent cannot be used after 48 hours.
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EXPERIMENT 4
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PART IV: SAFETY REGULATIONS IN
LABORATORY
1. Safety in the laboratory
You have to comply with safety regulations in laboratory
Be careful with chemicals used in the lab due to their high toxicity, irritation,
corrosion or flammability.
Be careful with fragile, glass, sharp and potentially infectious equipment,
material or sample such as disposable pipets, needles, blood, bacteria…
Waste chemicals, disposable equipment, and infectious agents must be
separated and discarded in suitable place in order to protect the working
condition in lab, public place and environment.
The electrical equipment, including hot plates, stirring motors, and high-
voltage power supplier present special hazards.
The accident may happen easily to anybody who do not follows the lab safety
regulations.
Using protective eyewear and gloves is highly recommended when working
with hazardous chemicals or infectious agents
Do not work alone in the laboratory.
Understand properties of all chemicals to be used such as their flammability,
reactivity, toxicity, and proper disposal.
Eating, drinking, and smoking are not allowed in laboratory.
Unauthorized experiments are not allowed.
Mouth suction should not be used to fill pipets or to start a siphons.
Be familiar with the location and use of standard safety features in your
laboratory.
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2. The laboratory notebook and experiment reports
The most readable notebook is one in which only the right hand pages are used
for record keeping. The left hand pages may be used for your own notes,
reminders, and calculation.
In the biochemistry lab, you must read the material very well at home
and write down the procedure in your notebook. This will be checked by
the lab instructor. If not, you will be asked to stop working and go out.
You must clean the lab-glassware carefully and return them to their previous
place because:
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Glass cuvettes: never clean cuvettes or optically polished glassware with
ethanol, KOH, or other strong base, as this will cause etching. All cuvettes
should be cleaned carefully with 0.5% detergent solution, in a sonicator bath,
or in a cuvette washer.
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References:
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