Study of Small scale disruption systems

Experiment No.: Date:

Aim:
To study the various small scale (laboratory) disruption methods and its comparison and to
measure disruption coefficient in small scale disruption methods

Principle:
The disruption of the cells is an important stage in the isolation and preparation of intracellular
product. Cell disruption techniques are classified into mechanical and non-mechanical methods.
Mechanical methods include homogenization, bead mill, and Jet stream methods.

 Mechanical Methods: Mechanical disruption

 Chemical Methods: Use of Detergent, Solvents, and autolysis
 Enzymatic methods: Lysozyme autolysis.

Homogenizer: A high-pressure homogenizer consists of high-pressure pump and cylinder. The

required pressure can be set manually. The cell suspension is brought to the pressure set by
means of pressure pump. The valve is open by about 0.075mm and the pressure rises above
that cell. This causes a sudden and very rapid drop in pressure of the cell suspension, which
rushes at approximately 350m/s through the orifice. The resulting turbulence, the shearing
forces and the direct collision with the pressure ring positioned at right angles to the direction
of flow, results in the disruption of the cell walls.
Lysozyme: Lysozyme cleaves the bond in the peptidoglycan layer of the cells and allows the
disrupted cells to ooze out.
Sodium Dodecyl Sulphate(SDS): Detergents are a class of molecules whose unique properties
enable manipulation of hydrophilic interaction of biomolecules. Detergents solubilize the
membrane protein and lipids of cell wall and release the soluble protein.

Materials required:
 Cells of Saccharomyces cerevisiae
 Lysozyme, Sodium Dodecyl Sulphate, CTAB
 Lowry’s reagent, FC reagent
 Microfuge tubes
 Centrifuge
 Spectrophotometer
Procedure:
 Commercially available baker’s yeast (Saccharomyces cerevisiae) is used for this
study. The overnight culture suspension with suitable dilution will be used separately
for each method.
 The initial cell OD is measure prior to treatments
 The cells are diluted in 1:1 ratio in distilled water
 SDS and CTAB are added at 0.2 M concentration to respective test tubes
 While lysozyme of volume 100 µL was added directly from the stock
  All the samples were mixed using a glass rod and incubated at room temperature for 20
minutes
 Post incubation the samples were centrifuged at 8000 rpm for 10 minutes
 Cell concentration was found before and after incubation(@ 600 nm)
 200 µL of the supernatant of sample was used to perform Lowry’s assay for protein
estimation
 2 ml of Lowry’s reagent was added and incubated for 10 minutes. Then 0.2 ml of FC
reagent(1:1) was added and incubated for 30 minutes
 The optical density of each sample was measured @540 nm

Method 1: Mechanical
A suspension of Saccharomyces cerevisiae was disrupted in a One Shot cell homogeniser with
volume of 10 ml and the rate of disruption determined by measuring the activity of the
Invertase, Fumarase and the amount of protein released over a period of time.
The optimal conditions for disruption will be determined by varying disruption pressure from
25 Kpsi – 40 Kpsi. The lysate is then centrifuged at 10000 rpm for 5 mins to sediment the
cellular debris and the un-lysed cells. The pellet will be diluted with suitable volume of
distilled water and measured for cell concentration, Total protein amount and enzyme activity.
Method 2: Chemical
The cell suspension is diluted with distilled water and then SDS buffer added with varying
concentration from 2- 5 % W/V. The mixture is allowed to incubate at room temperature for 20
minutes and at centrifuged at 10,000 rpm for 5 minutes. After centrifugation, the supernatant is
transferred to microfuge tube and the measured for cell concentration, Total protein amount
and enzyme activity.
Method 3: Enzymatic
The cell suspension is diluted with distilled water and then Lysis buffer added with varying
concentration. The mixture is allowed to incubate at room temperature for 20 minutes and
centrifuged at 10,000 rpm for 5 minutes. After centrifugation, the supernatant is transferred to
microfuge tube and the measured for cell concentration, Total protein amount and enzyme
activity.
Observations:
1. Cell concentration(Absorbance @ 600nm) initially = C0 =C1=
2. Cell concentration after treatment=C
3. Total protein concentration(Absorbance @ 580nm)
4. Volume of protein sample after each treatment= 1 ml
5. Volume of sample taken for Lowry’s assay= 0.2 ml
6. OD of Control(Extracellular protein)
7. Concentration of protein=OD/(Slope of protein calibration curve)
8. Amount of intracellular protein conc.=Amount of protein in supernatant-Control conc.
9. Cell disruption constant K = e(C1-C) for each treatment-SDS,CTAB, Lysozyme
(K= Batch cell disruption constant; C1=Initial cell conc.; C=Cell OD after incubation )
Results:
 Cell Disruption coefficient Optimization

Experiment No.: Date:

Aim:
Determination and optimization of the cell disruption coefficient for the given cell suspension
using chemical solubilization method.
Principle:
The disruption of the cells is an important stage in the isolation and preparation of intracellular
product. Cell disruption techniques are classified into mechanical and non- mechanical methods.
Mechanical methods include homogenization, bead mill, and Jet stream methods. Non-mechanical
methods are further classified into-
Chemical Methods: Use of detergent, solvents, and autolysis
Enzymatic Methods: Lysozyme autolysis.
Materials required:
 Cells of Saccharomyces cerevisiae
 Sodium Dodecyl Sulphate, CTAB, Lysozyme
 Lowry’s reagent, FC reagent, DNS
 Microfuge tubes
 Centrifuge
 Spectrophotometer
Procedure:
1. Commercially available baker’s yeast (Saccharomyces cerevisiae) is used for this study.
2. The overnight culture suspension with suitable dilution is used separately for each method.
3. The initial cell OD is measure prior to treatments
4. The cells are diluted in 1:1 ratio in distilled water
5. SDS and CTAB are added at different concentrations [0.1,0.2,0.3 M] to the respective
test tubes
6. While lysozyme of different volumes [80,100,120 µL] was added directly from the
stock to different test tubes
7. All the samples were mixed using a glass rod and incubated at room temperature for 20
minutes
8. Post incubation the samples were centrifuged at 8000 rpm for 10 minutes
9. Cell concentration was found before and after incubation(@ 600 nm)
10. 200 µL of the supernatant of sample was used to perform Lowry’s assay for protein
estimation
11. 2 ml of Lowry’s reagent was added and incubated for 10 minutes. Then 0.2 ml of FC
reagent(1:1) was added and incubated for 30 minutes
12. The optical density of each sample was measured @540 nm
13. The samples were also used for DNS assay to determine the protein activity
Observation:
1. Cell concentration(Absorbance @ 545nm) initially = C0 =C1=
2. Cell concentration after treatment=C
3. Total protein concentration(Absorbance @ 580nm)
4. Cell disruption constant K = e(C1-C) for each treatment-SDS,CTAB, Lysozyme
(K= Batch cell disruption constant; C1=Initial cell conc.; C=Cell OD after incubation )
5. Protein Assay:
 Volume of protein sample after each treatment= 1 ml
 Volume of sample taken for Lowry’s assay= 0.2 ml
 OD of Control(Extracellular protein)=
 Concentration of protein=OD/(Slope of protein calibration curve)
 Amount of intracellular protein conc.=Amount of protein in supernatant-Control
conc.
6. DNS Assay:
 Volume of protein sample after each treatment= 1 ml
 Volume of sample taken for DNS assay= 1 ml
 OD of Control(Extracellular protein)=
 Glucose conc.=OD/(Slope of Glucose standard curve) (mg/ml)
 Reaction rate=Glucose conc./Reaction time (mg/ml min)
 Enzyme activity=Reaction rate/(Mol. Wt. of Glucose) (IU/ml)

Results:
Serial Method Treatment Cell OD Enzyme Protein Disruption
No. conc./Volume activity conc. Coefficient
K

1 SDS

2 CTAB

3 Lysozyme
 Membrane filtration- flux determination in cross flow filtration

Experiment No.: Date:

Aim:

To harvest Saccharomyces Cerevisiae cells using cross flow membrane filtration and its flux
studies.
 Optimization of flow rate for feed filtration
 Flux curve construction-volume collected over time

Principle:

Cross flow is an alternative method to classical solid liquid filtration (Dead-end filtration).
Parallel flow across the membrane placed at proper angles to the direction of filtration, to
preclude the build-up of a filter ‘cake’. Ultra-filter or micro pore membranes are used as the
filter material. The pressure across the membrane is defined as trans-membrane pressure
(TMP). Under ideal conditions, the resistance to filtration in CFF is dependent only on the
filter material used. This experiment, harvesting of cells is carried out in the filter module of
0.026 m2 surface area & a pore diameter of 0-2 micrometre. A cell suspension is pumped at
high speed through the module so that it crosses the membrane at right angles to the direction
of flow. The material retained and filtered is returned to the reservoir tank and the filtrate
(permeate) and collected separately. Transmembrane pressure (TMP), the force responsible for
the filtration can be regulated by adjusting the pressure at the entry and exit of the cells
suspension.

Material required:
1. Yeast culture- S.cerevisiae, 24 hrs incubated culture (200 ml)
2. Cell should be suspended in potassium phosphate buffer (50mM), pH adjusted with 0.1M
KOH to pH 7.0(optional)
3. Mid Gee filtration system contains feed reservoir; permeate collector; peristaltic pump
4. Spectrophotometer
5. Distilled water
Crossflow filtration-Schematic Flow path:

Procedure:

1. Before switching on the pump connections of the membrane tubing’s (to and from the
reservoir tank) are checked.
2. Water is initially filled in the reservoir and pump is switched on, filtrate is collected and
measured at equal time intervals.
3. The yeast suspension is pumped through the membrane and the filtrate collection
vessels tubing connections are opened slowly.
4. Graph was plotted denoting the water flux (standard and the membrane flux (sample)
and the flux was calculated from the data.
5. The TMP is fixed at a particular point y calculating the flux. Optical Density is measured
at 600 nm and the dry weight of yeast is determined at every 50 ml of filtrate obtained.
6. The membrane is regenerated by washing with 0.1M NaOH buffer/ 40OC warm water
The values for flux, O.D. and dry weight are plotted against the volume of the filtrate.
Observation:
1. Culture dilution= 20ml overnight+130 ml distilled water
2. Initial culture OD @ 600nm (Ao)=
3. Transmembrane Pressure = Pressure entry point + Pressure exit point - Filtrate pressure
2
4. Flux = Volumetric flow rate / Area of Membrane , Lt/ min.m2

Results:
1. Volume of filtrate Vs Flux
2. Volume of filtrate Vs Cell Concentration
 Egg white isolate fractionation using ammonium sulphate precipitation

Experiment No.: Date:

Aim:
Partial purification of egg white isolate using ammonium sulphate saturation.

Principle:
The protein precipitation obtained at high concentrations of ammonium sulphate is the result of
a decrease in the hydration of proteins in favour of that of the ion of the neutral salt. The
hydration layer surrounding the surface of the protein. The concentration required to precipitate
a specific protein is dependent on the salt used and is also a function of pH and temperature. The
reproducibility of a protein precipitation is also dependant on the rate at which the salt is added
[30-80 %], the length of time the suspension is stirred and whether the salt is added as a finely
ground powder or as a saturated solution of ammonium sulphate. The use of a saturated solution
of ammonium sulphate is gentler on the protein but is only to be recommended for small
volumes since the volume of the sample doubles when 50% saturation is reached.

Materials:
 Chemicals: Finely ground ammonium sulphat
 Centrifuge
 Magnetic stirrer
 Stop watch
 pH meter.
 Sample: Egg white
 Distilled water
 Microfuge tubes
(% saturation of ammonium sulphate (A)= Amount in grams of ammonium sulphate to be
added to 1 litre of protein solution(B))

Procedure:
1. The extracted egg white is collected in a clean beaker and made up to 100 ml using
distilled water
2. An ammonium sulphate precipitation with the following degrees of saturation (at room
temperature) is to be performed: 30,40,50,60,70 and 80%.
3. pH of the albumin solution prior to salt addition is measured
4. After the addition on salt, the mixture is vigorously stirred in a magnetic stirrer
5. For each conc. Of salt, the pH of mixture is noted and sampling is done periodically
6. The final volume is measured before determining the protein concentration and
proceeding to the next salt conc.
7. For the successive conc. The difference in amount of salt required is added
8. All the samples collected are spun at 8000 rpm for 10 minutes
9. Supernatant thus obtained is added to clean tubes
10. This supernatant is used for protein estimation
11. Salt free albumin extract was used as the control
Observation:
Salting out point for Ammonium Sulphate(Equilibrium conc.)= 4.1 M
Molecular weight of Ammonium Sulphate= 132 g/g mol
OD of samples were taken @600 nm
Volume of protein sample after each treatment= 2 ml
Volume of sample taken for Lowry’s assay= 0.2 ml
Concentration of protein=OD/(Slope of protein calibration curve)

(For 0.2 ml of sample)

Seria Conc Volume of Volume Amount OD Amount of Amount of
l No. . of mixture(ml after of salt @60 protein in protein
salt ) sampling(ml added(g 0 nm supernatant(µg precipitated(µg
(%) ) ) ) )

Concentration of salt added to mixture(%) Protein precipitated(µg) for 2 ml of sample

taken

Results:
The optimal percentage of ammonium sulphate saturation at which maximum protein precipitation
was found at
 Separation of casein from milk by Isoelectric point method

Experiment No.: Date:

Aim:
To isolate casein from milk by isoelectric precipitation and to estimate the percentage of casein
precipitated.

Principle:
The isoelectric point (pI) of a protein is the pH where the net charge of the protein is zero.
Proteins tend to aggregate and precipitate at their pI because there is no electrostatic repulsion
keeping them apart. Proteins have different isoelectric points because of their different amino
acid sequences (i.e. relative numbers of amino acid anionic and cationic groups), and thus can
be separated by adjusting the pH of a solution.

When microorganisms grow in milk, they often produce acids and lower the pH of the milk.
The phenomenon of precipitation or coagulation of milk protein (protein) at low pH as milk
becomes spoiled is one of the common examples of protein isolation due to changes in the pH.

Materials Required:
 Milk(Low fat)
 Glacial acetic acid (0.2M)
 Lowry’s Reagent
 0.2 mg/ml BSA
 pH meter
 Microfuge tubes
 Pipette, tips
 Centrifuge

Procedure:
1. Prior to using the semi-pastuerized milk, the milk is spun at 8000 rpm for 5 to 10 minutes to
remove the fat content(as it interferes with protein estimation).
2. pH of raw milk is determined using a pH metre (with appropriate dilution)
3. 25ml of undiluted milk and 25 ml of diluted(1:1) milk is taken in separate conical flasks
4. 2ml from each is kept separately to serve as a control prior to acid addition
5. 0.2 M acetic acid is added slowly using a pipette to it until precipitation occurs. The volume
of acetic acid is noted.
6. pH of milk solution is determined.
7. 1 ml of this solution is taken in an microfuge tube and centrifuge & at 10000 rpm for 5 min.
8. The protein content in the supernatant is estimated by Lowry’s method
9. The extraction efficiency is calculate in each case
Observation:
Isoelectric point of casein= 4.2(pH)
Initial pH of diluted and undiluted milk sample= 6.2

Dilution Amount of Actual amount

Sample Factor OD at 600nm protein (µg) of protein(µg)
Raw milk-
undiluted 1:0
Raw milk-
undiluted
(No acid-
control) 1:0 -
Diluted milk
1:1
Diluted milk -
(No acid- 1:1
control)

Calculations:

A) For diluted milk sample

Amount of protein=

Percentage of protein recovery=

B) For undiluted milk sample

Amount of protein=

Percentage of protein recovery=

Result
 Protein partitioning by Aqueous Two Phase extraction(ATP)

Experiment No.: Date:

Aim:
To separate the major protein in the given extract by two phase method. Thereby, to optimize,
the ATP mixture composition for that particular protein extraction.

Principle:
Aqueous two phase extraction is a technique used in biochemistry profession and, to a
lesser extent, in the biotechnology industry, to isolate and purify a variety of proteins and
biological materials including sub cellular particles and whole cells. This technique was
developed to its present form by Albertsson.

Two phase aqueous systems are made by combining two incompatible water soluble
components (ex: IL and Salt) or a polymer and a salt in water, above a critical
concentration. This causes the system to separate into two immiscible phases and the
proteins partition between the two phases. Recent advancements has brought in greener
separation methodology based on Ionic liquids. These are usually organic salts which are
normally liquid at room temperature. RTILs, when mixed with an appropriate stock
solution of ammonium sulphate, would form two separate layers (phases) due to the
reduction in entropy. Tetrabutyl phosponium bromide/Ammonium sulphate system is
suitable for protein extraction with-out any harmful effects on the protein. The partition
coefficients of different ATP systems can be manipulated in this system by the addition
of acid, base, partitioning ions, no partitioning ions, and surfactants.

Physical measurements:
In a single-stage extraction, a salt solution along with a certain amount of protein is
thoroughly mixed with a solution of PEG. Protein partitions between the two phases until
equilibrium is reached, the equilibrium partition coefficient k is defined as
K= Y / X
Where Y = Concentration of protein in the extract phase
X = Concentration of protein in the raffinate phase
The objectives of this experiment are as follows:
1. To show that the PEG/Salt mixture forms two phases above certain concentrations.
2. To estimate phase volume ratios.
3. To determine the equilibrium partition coefficient of a protein.
4. To determine the yield of the product.
Materials:
 Salt(Ammonium sulphate, Sodium sulphite, Potassium sulphate)
 PEG(Polyethylene Glycol) of varying molecular weights
 Sample(Pineapple, Ginger, latex)
 Glass rod, test tubes, microfuge tubes
 Lowry’s reagent, FC reagent
 Distilled water
 Centrifuge
Procedure:
1. The following aqueous stock solutions are used.
a. 20% PEG- MW (4000,6000,8000)
b. 15% (w/w) salt solution
2. Formulation of the phase system:
a. A 15 ml capacity test tube is taken and 10 ml of samples were taken separately
b. The appropriate amounts of PEG, salt stock solutions are added carefully to the tube
3. Each phase system is vortexed. Dissolution of all the salts in the mixture is made sure. The
tube is shook several times to help in dissolving the salt.
4. The phase systems in all tubes are kept still at room temperature to allow its separation into
top and bottom phases
4. Each system is centrifuged at 6000 rpm for ten minutes to separate the two phases(if
required)
5. The top and bottom phases are separated into different tubes and their volume is measured
6. The phases are removed separately taking care not to disturb the interphase.
7. The concentration of the protein in each phase is determined using a spectrophotometer by
Lowry’s method for protein estimation
8. 0.2 ml of sample is taken for Lowry’s assay
9. The two phases from each sample at different PEG/salt systems are also subject to protease
assay to determine the enzyme activity
10. 2 ml of sample is taken for protease assay and to this 1 ml of casein dissolved in 0.1 N of
NaOH solution is added
11. Samples are incubated for 10 minutes and the reaction is stopped by adding 10%
TCA(Trichloro acetic acid)
12. These samples are then subject to Lowry’s assay to measure remnant protein
Observation:
1. Enzyme partition coefficient k = Ct/ Cb
2. Phase volume ratio: V=Vt/Vb
Where Vt and Vb=Volume at top and bottom phase respectively
3. Purity factor: B=Ct/ CO
B= Cb/CO
Where, Ct and Cb = Protein concentration at top and bottom phase respectively
4. Yield:
 Top phase=(kV)/(1+kV)
 Bottom phase=1/(1+kV)
5. Enzyme activity: (Absorbance @600 nm/Reaction time) X (1/Reaction volume)
6. Control= 3 ml water+15% salt+ 20% polymer(PEG)
Blank= 0.2 ml water+ 2 ml Lowry’s reagent + 0.2 ml FC reagent

Results:
 Protein partitioning by Reverse Micellar Extraction

Experiment No.: Date:

Aim:
To study the protease partitioning from natural sources by reverse micellar extraction
using CTAB and SDS.

Principle:
Reverse micelles are thermodynamically stable nanometre-sized water droplets within an
organic solvent stabilised by a monolayer of surfactant molecules and can be formed by
contacting an aqueous phase with an immiscible organic phase containing these
surfactants. Their inner cores contain an aqueous microphase which is able to solubilise
bioproducts such as amino acids and proteins. It has been reported that the protein is still
active inside the core, and can be reextracted into an aqueous phase under certain
conditions of pH and ionic strength.

Materials:
 0.2M CTAB and 0.2M SDS
 Isooctane
 50 mM NaOH to adjust pH
 0.5 M potassium chloride
 Samples(Pineapple, Ginger, latex)

Procedure:
A) Preparation of sample:
1. Collect fresh juicy ginger and remove the skin.
2. Cut it into equal and small pieces
3. Weigh a known amount of buffer and add it to the mortar and pestle
4. Add a small quantity of buffer (phosphate) and grind the ginger piece to extract the
protease enzyme.
5. Filter the product to obtain a clear crude enzyme extract
B) CTAB or SDS/Iso octane/system:
 Forward Extraction :
1. Take 5 ml of crude enzyme extract and adjust the pH of the sample to 8 and
add 0.5 M KCl which serves as aqueous phase of reverse micellar system.
2. For reverse micellar phase add 0.2M CTAB/SDS, 5ml of Isooctane.
3. Mix both phase and vortex for 10 min.
4. Centrifuge the mixture for 10min at 3000 rpm.
5. Or allow it to separate into two layers by incubating it at room temperature for
30 minutes
 Back ward Extraction:
1. Take 2ml of reverse micellar phase from forward extraction.
2. Add 2ml of fresh aqueous phase of pH 4 containing 0.5 M of KCl in Distilled
water.
3. Vortex the mixture for 10 min and centrifuge it for 10 min at 3000rpm
4. Take the aqueous phase and check for protein concentration and Enzyme
activity.
 C) Lowry’s assay:
1. The concentration of the protein in each phase is determined using a
spectrophotometer by Lowry’s method for protein estimation
2. To 0.2 ml of sample, 2ml of Lowry’s solution is added and incubated for 10
minutes
3. Following this 0.2 ml of FC reagent is added and incubated at room
temperature for 30 minutes (the test tubes are shook to mix the contents
thoroughly
4. OD is measured at 600 nm

D) Protease assay:
1. The two phases from each sample at different PEG/salt systems are also subject to
protease assay to determine the enzyme activity
2. 2 ml of sample is taken for protease assay and to this 1 ml of casein dissolved in
0.1 N of NaOH solution is added
3. Samples are incubated for 10 minutes and the reaction is stopped by adding 10%
TCA(Trichloro acetic acid)
4. These samples are then subject to Lowry’s assay to measure remnant protein
Observation :
Control= 5 ml water+ 0.2M detergent(SDS or CTAB)+ 5 ml isooctane
Blank= 0.2 ml water+ 2 ml Lowry’s reagent + 0.2 ml FC reagent

Serial No. Sample OD for Lowry’s OD for Protease assay

assay
1 CTAB Ginger(T)
2 CTAB Ginger(B)
3 CTAB Pine(T)
4 CTAB Pine(B)
5 CTAB Latex(T)
6 CTAB Latex(B)
7 CTAB Control(T)
8 CTAB Control(B)
9 SDS Ginger(T)
10 SDS Ginger(B)
11 SDS Pine(T)
12 SDS Pine(B)
13 SDS Latex(T)
14 SDS Latex(B)
15 SDS Control(T)
16 SDS control(B)

Results:
 Desalting of protein by Diafiltration

Experiment No.: Date:

Aim:
Following precipitation with neutral salt or column chromatography, protein exists in
solutions with a high salt concentration. For later purification steps the protein solution
has to be conditioned i.e. the amount of salt reduced and possibly placed in another
buffer system. On a small scale, this can be done in dialysis tubing. For larger volumes
gel filtration or filter dialysis allow efficient desalting and change of buffer. To find out
the wash volume required to minimize the ionic strength of the given sample (Protein
suspension).

Principle:
In diafiltration, membranes are used which are impermeable to protein molecule but do
not hinder the passage of smaller molecule. Membranes are particularly useful for the
exchange of salts or sugars or for the removal of low molecular weight substances; the
ionic strength and the pH of the solution can be altered at the same time. The rate of
desalting is the same as the filtration rate because the salt transportation is a convection
process. The rate of desalting is only a function of ultrafiltration rate and is independent
of the concentration of the substances being separated. The filter dialysis performed here
is done in a cross flow module. The aim of cross filtration is, by appropriate flow
mechanics, e.g. parallel flow across the membrane combined with an effective
intermittent gradient acting at right angles to the direction of filtration, to prevent the
built up of a secondary membrane of protein.

Materials:
 Protein Solution: It is possible to use a particle free crude extract, the
resuspended pellet from an ammonium sulphate precipitation, or filter dialysate
from a cell homogenate.
 Apparatus: AMERSHAM cross flow filter system
 Buffer: 50mM potassium phosphate, pH 7.5, a similar buffer of low morality or
demonized water should be used for filtration dialysis.
 Conductivity measurement: The conductivity meter should be coupled to a
measuring device allowing the conductivity to be measured continually.
Procedure:
1. Ammonium sulphate or sodium chloride is added to 100ml protein solution until the
conductivity of the solution has a value of about 100mS/cm. The cross flow filtration
is performed in a AMERSHAM system. (Midgee filter module with a filter area of
0.0026 cm2; Cut off 10 KDa). The pump should deliver about 40 L/hour and the
pressure across the membrane should lie between 0.8 to 1.0 bar.
2. The device for measuring the conductivity is placed in the high salt protein solution
so that the reduction in conductivity (ion concentration) can be monitored
continuously throughout the filtration.
3. The high salt protein solution is maintained at a constant volume by the addition of
fresh buffer to compensate for the loss generated by the production of filtrate.
4. Throughout the filter dialysis, the flux should be determined as a function of time: this
is done by measuring the time required to collect 20ml filtrate. The filter dialysis is
continued for as long as is necessary to reach the desired conductivity.
5. The volume of buffer required is at least six times that of the protein solution. The
 graph illustrates the course of a typical cross flow filter dialysis.

Observation:

Volume of filtrate(ml) Time of filtration(sec) Conductivity (ms/cm)

Results:
 PEG-Assisted precipitation of protease

Experiment No.: Date:

Aim:
To purify ovomucin by PEG assisted hydrotropic precipitation

Principle:
PEGylation is the process of attachment of PEG molecule to a macromolecule like protein to
increase its molecular weight and biological activity. Recent trend in green separation
involves co-precipitation of proteins from various sources obtained at varied concentrations
of polyethylene glycol which yields greater purity without loss of any biological activity. The
polymer binds to specific sites in the surface of the protein. The concentration required to
precipitate a specific protein is dependent on its molecular weight. PEG conjugation masks
the protein's surface and increases the molecular size of the polypeptide. The reproducibility
of a protein precipitation is also dependant on the percentage of polymer added, molecular
weight of protein and polymer.

Materials required:
 Source: Separated egg white.
 Chemicals: Polyethylene glycol (various molecular weight and percentage)
 Refrigerated centrifuge; Magnetic stirrer; stop watch.

Procedure:
1. A known mass of egg white was taken and 3 volumes (w/v) of 50mMNaCl
solution was added with continuous stirring for 2h to attain the equilibrium.
2. The pH is measured and adjusted to 6.0 with 2M HCL. Following, 3% (w/w)
PEG was added and the dispersion was allowed to sit for 2h.
3. Further, the homogenate was collected and centrifuged at 10000 rpm.
4. The pellet is taken up in 50 ml 0.5 M Sodium chloride buffer for removing
impurities, stirred for 4h and then centrifuged.
5. The pellet is collected and suspended in Tris-HCl buffer (pH 8.0, 20 mM)and
total protein concentration is determined.
6. For comparative purposes, these measurements must also be made for the crude
extract before the PEG precipitation (100% values).
7. Different molecular weight of PEG plays an important role in recovery amount.
Hence, PEG solutions of different molecular weight are prepared and recovery is
noted.
8. The values for the enzyme activities and protein concentrations depend on the
volume and must be multiplied by the volume to calculate the total amount.
9. Precipitate checked for purity by measuring the recovery at 280 nm and %
recovery of protein from total protein in egg white is estimated.
 Observation:

Amount of Total protein

Molecular Total protein in
protein content before % Recovered
Weight precipitate
precipitated Precipitation
PEG (mg/ml)
mg/g of EW mg/ml

4000
6000
8000
10000

Results:
The optimal condition found suitable for the maximum protein fractionation found at:
 Microwave assisted extraction of Protease

Experiment No.: Date:

Aim:
 To isolate Protease enzyme using microwave assisted extraction.
 Optimize the time for extraction

Principle:
Microwave assisted extraction is a modern technique used, in the biotechnology industry
to extract a variety of compounds and biological materials including sub cellular
particles and plant metabolites and is regarded as greener extraction process.

MAE is a recently developing technique which has pioneer applications in biotechnology

research field like Microwave assisted ATPS, microwave assisted synthesis. Recent
advancements have brought in greener extraction methodology based on microwave to
replace existing methodology of Soxhlet extraction with improved efficiency.
Microwave assisted extraction would recover the product in more effective way and with
less usage of solvent system. Microwave can be customised as per requirement for any
kind of microwave assisted process.

In a single-stage extraction, the source is soaked in a suitable solvent system and

microwave is applied. Samples were collected at different time interval and using
different solvent percentage. These were estimated for their protease content by Lowry’s
assay and Protease assay. The objectives of this experiment are as follows:
 To assist efficient extraction of protease from sample with microwave
 To estimate the recovery
 To determine the optimal conditions of recovery.
Procedure:
A) Preparation of sample
1. Collect fresh sample and wash them with water.
2. Soak the sample in Luke warm phosphate buffer (pH 8.0) for 2 hours.
B) Extraction by conventional method with solvents
1. Weigh the desired amount of seed
2. Crush the seeds and add equal volume of phosphate buffer(w/v)
3. Centrifuge the content @ 6000 rpm and collect the supernatant
4. Record the absorbance by setting λ at 502 nm
C) Formulation of extraction mechanism in MW:
1. Carefully weigh the seed and note down the weight.
2. Add phosphate buffer till the seeds get soaked .
3. The vessel is placed symmetrically in the microwave field.
4. Samples were collected at various time intervals at same temperature.
5. Further, temperature was varied and samples were collected at same interval.
6. Finally, trials were taken with usage of different solvent .
7. Measure the optical absorption in the spectrophotometer.
8. Determine the concentration of the protein in each phase using a spectrophotometer.
9. Set wavelength λ at 502 nm.
10. Use pure water to set blank.
11. Record each sample.

Observation:
1. Extraction efficiency k = Absorbance in mw assisted / Absorbance in solvent assisted
2. Yield maximised with low volume of solvent
Results:
 Liquid chromatography

Experiment No.: Date:

Aim:
To purify the enzymes Glucose-6-phosphate dehydrogenase and fumerase from the
Saccharomyces cerevisiae broth using an Ion Exchange Chromatography (IEX).
Principle:
Ion exchange chromatography works on the basic principle that oppositely charge particles
are attracted to each other. The stationary phase consists of fixed charges on a solid support.
These charges can be either negative or positive. Hence there are two types of ion exchanger
i.e., cation and anion exchangers.
Cation exchangers possess negatively charged groups and attract positively charge molecules,
e.g. caboxy-methylo cellulose or CM-cellulose. Conversely, anion exchangers have
positively charged groups that attract negatively charged molecules and thus separate anionic
molecules. E.g. Diethylaminoethyl-cellulose.
Proteins are complex ampholytes i.e., they have both positive and negative charges and can
be separated from a mixture of compounds on the basis of net positive or negative charge that
carry. Isoelectric point of a protein (pl) is the pH at which its net charge is zero (i.e., number
of positive and negative charges are equal). Therefore, proteins will have either a net negative
charge or net positive charge depending on the pH of the solution and thus, it is possible to
use either an anion exchanger or a cation exchanger.
In ion exchange chromatography, solution containing protein of interest is applied to the ion
exchanger. Protein binding to the ion exchanger is dependent on the net charge of the protein
at that particular pH and on the ionic strength of the mobile phase. Bound protein is then
eluted out from the stationary phase by increasing the concentration of the counter ions or by
changing pH, which alters the charge on the protein. Weakly charged protein is displaced
from the stationary phase with lower concentration of the counter ions than highly charged
protein. This result in the separation of the protein based upon its net charges.
Extent of purification of protein can be determined by comparing its specific activity.
Specific activity is the ratio of enzyme activity to mass of the protein in the sample, usually
expressed as units of activity per milligram of protein (U/mg). As the enzyme is purified
(through a no of steps) other proteins in the mixture are eliminated while most of the enzyme
activity is retained, this results are increase in the specific activity of the enzyme. Hence, by
determining so specific activity before and after purification, one can determine the fold
purification and yield of the enzyme.
Materials:
 Microorganism: Saccharomyces cerevisiae
 Equipment: Akta Prime LIQUID CHROMOTOGRAPHY SYSTEM Column
specification: Q – sepharose FF from GE (strong basic anion exchanger)

Procedure:
1. High purity water and chemicals are used and all buffers are filtered through
0.045um filter(before use)
2. The instructions are read carefully before running the system and the manufacturers
suggestions are followed for better result
3. Preparing the buffers
a) Start buffer (port A1): 20mM Tris-HCl, pH-8 Elution buffer port B: 20mM Tris-
HCl,1.0 M NaCl, pH-80 Prepare at least 500 ml of each buffer
b) Preparing the sample
4. The sample is adjusted to composition of start buffer by:
i) Diluting the sample in start buffer
ii) Pass the sample through a 0.45 µm filter.
5. Preparing the system
a) The inlet tubing is placed from port A1 (8-port valve) in the binding buffer and
the tubing from port B (port valve) in the elution buffer.
b) The three brown waste tubing is placed in waste.
c) The column is connected between port 1 on the injection valve (7-port valve) and
the UV flow cell.
d) The fraction collector rock is filled with 18 mm tubes and position the white plate
on the fractionation arm against the first tube.
 e) A sample loop is connected which is large enough for the sample between port 2
and 6 on the injection valve. Use a syringe to manually fill the loop.
f) Selecting application template and starting the method
g) Check the communication to Prime view. At the lower right corner of the screen,
the Controlled by prime should be displayed.
h) Use the arrow and ok buttons to move in the menu tree until you find Anion
Exchange HiTrap Q.
i) Enter the sample volume and press ok to start the template.
Observation:
Model Chromatogram:

Determination of fumerase activity. 25ml 1M potassium phosphate buffer is added to 3.352

g/l malic acid and the pH adjusted to 7.3 with 1M KOH. The volume is made up to 500ml
with demonized water. This is the stock solution which can be stored frozen in small aliquots.
Enzyme assay: stock solution 1000μl; enzyme solution (may be diluted if necessary) 50μl.
The test is performed at 30oC and the change in extinction at 250 nm is measured (coefficient
of extinction: 1.45 cm2 / μ Mol).
Results:
Sample: protein mix containing G-6-PDH and fumerase in start buffer
Appendix:1

Total Protein Estimation by Lowry’s Method

Aim:
To construct the calibration curve of proteins using BSA as standard by Lowry’s method.
Principle:
The phenolic group of tyrosine and tryptophan residues (amino acid) in a protein will produce
a blue purple color complex, with maximum absorption in the region of 660 nm wavelength,
with Folin- Ciocalteau reagent which consists of sodium tungstate molybdate and phosphate.
Thus the intensity of color depends on the amount of these aromatic amino acids present and
will thus vary for different proteins. Most proteins estimation techniques use Bovine Serum
Albumin (BSA) universally as a standard protein, because of its low cost, high purity, and
ready availability. The method is sensitive down to about 10 μg/ml and is probably the most
widely used protein assay despite its being only a relative method, subject to interference
from Tris buffer, EDTA, non-ionic and cationic detergents, carbohydrate, lipids, and some
salts. The incubation time is very critical for a reproducible assay. The reaction is also
dependent on pH and a working range of pH 9 to 10.5 is essential.
Reagents required:
 BSA stock solution (1mg/ml)
 Analytical reagents:
a) 50 ml of 2% sodium carbonate mixed with 50 ml of 0.1 N NaOH solution (0.4 gm
in 100 ml distilled water.
b) 10 ml of 1.56% copper sulphate solution mixed with 10 ml of 2.37% sodium
potassium tartarate solution. Prepare analytical reagents by mixing 2 ml of (b)
with 100 ml of (a)
 Folin - Ciocalteau reagent solution (1N) Dilute commercial reagent (2N) with an
equal volume of water on the day of use (2 ml of commercial reagent + 2 ml distilled
water)
Procedure:
1. Set up eleven sets of three 16 x 150 mm test tubes in rack.
2. Add BSA [1,2,3,4,5 ml] corresponding to a concentration of [0.2,0.4,0.6,0.8,1 µg/ml]
to these tubes
3. The volume in each tube is made up to 5ml by adding distilled water
 4. Add 2 ml of Lowry’s reagent to all the test tubes
5. Incubate for 10 minutes at room temperature.
6. Following the incubation add 0.2 ml of dilute Folin’s reagent(1:1) to each tube.
7. Gently mix the contents of the tubes
8. Incubate at room temperature for 30 minutes.
9. Measure the optical density @ 580nm.
10. The calibration chart is prepared for BSA at various concentrations from the
observations.

Observation:

Lowry’s
BSA Water Sample Conc. Sample Folin’s reagent O.D
reagent
(mL) (mL) (mg/mL) Volume (mL) (ml) at 600nm
(mL)
1 4 0.2 0.2 2 0.2
2 3 0.4 0.2 2 0.2
3 2 0.6 0.2 2 0.2
4 1 0.8 0.2 2 0.2
5 0 1.0 0.2 2 0.2

Report:

The standard curve was plotted and the slope of the standard graph for BSA was found to be
Appendix:2
Total reducing sugar analysis by DNS (3, 5-dinitro salicylic acid) method
Aim:
To construct calibration curve for total reducing sugars using Glucose as standard by DNS
method.
Principle:
This method tests for the presence of free carbonyl group (C=O), the so-called reducing
sugars.
This involves the oxidation of the aldehyde functional group present in, for example, the
ketone functional group in glucose and fructose. When alkaline solution of 3,5-
dinitrosalicylic acid reacts with reducing sugars (eg. Glucose, lactose) it is converted into 3-
amino-5-nitrosalicylic acid with orange color.

Because dissolved oxygen can interfere with glucose oxidation, sulphite, which itself is not
necessary for the color reaction, is added in the reagent to absorb the dissolved oxygen. The
above reaction scheme shows that one mole of sugar will react with one mole of 3, 5-
dinitrosalicylic acid. Different reducing sugars generally yield different color intensities;
thus, it is necessary to calibrate for each sugar. This is a convenient and relatively
inexpensive method owing to its relatively low specificity.
Reagents required:
 Glucose standard solution (g/L)
 DNS Reagent (Dinitro salicylic acid solution 1%)
a) Dinitro salicylic acid - 10 g
b) Phenol – 2 ml
c) Sodium sulphite - 0.5 g
d) Sodium hydroxide - 10 g
 e) Water – 1 litre
 Rochelle’s salt solution (Potassium sodium tartarate solution) - 40%

Procedure:
1. Different concentrations of Glucose with 0.1, 0.2, 0.3, 0.4 and 0.5 mg/mL were added
to test tubes from a stock solution of conc. 20 mg/mL
2. 3 mL of standard glucose solutions were taken in labelled test tubes.
3. Then, 3 mL of DNS reagent was added to each test tube and capped.
4. The mixture was maintained at 90ºC for 5-15 minutes in a hot water bath to develop
the red-brown color. And then it was cooled in an ice bath.
5. To the cooled mixture, 1 mL of Rochelle’s salt solution was added.
6. The absorbance was recorded at 540 nm in a Colorimeter/spectrophotometer.
7. 3 mL DNS solution + 3 mL distilled water mixture was used as a blank.

Observation:
Stock concentration of Glucose=20 mg/ml

DNS
Volume of Water Sample Conc. Sample Volume O.D
reagent
Glucose (ml) (mL) (mg/mL) (mL) at 540 nm
(mL)
0.6 2.4 0.1 3 3
1.2 1.8 0.2 3 3
1.8 1.2 0.3 3 3
2.4 0.6 0.4 3 3
3 0 0.5 3 3

Results:
The standard curve for glucose was plotted and the slope was found to be