21BTC402J Bio-separation Technology

Laboratory

SEMESTER- VII

YEAR: 2025-26

NAME:
REG NO:

SCHOOL OF BIOENGINEERING
DEPARTMENT OF BIOTECHNOLOGY
SRM INSTITUTE OF SCIENCE AND TECHNOLOGY
(Under Section 3 of UGC Act, 1956)
KATTANKULATHUR- 603 203.
 BONAFIDE CERTIFICATE

This is to certify that the record work done by -----------------------------------------

-------------------------------- , IV year /VII semester B.Tech., Biotechnology,

specialization in ------------------------------ for 21BTC402J Bioseparation Laboratory is

duly submitted for the end semester examination held on ----------------------------- in

the Department of Biotechnology, School of Bioengineering, SRM Institute of

Science and Technology, Kattankulathur – 603 203, during the academic year 2024-

2025.

Faculty In-charge Head of Department

Internal Examiner - I Internal Examiner - II

 INDEX

Name: Class:
Reg. No.: Branch:

Exp. Date of Page Date of

Title of Experiment Signature
No. Performance No. submission
Estimation of Protein by Lowry’s
1 method
Estimation of Protein by Bradford
2 method

3 Cell Disruption by Sonication

Cell Disruption by Enzymatic

4 Method
Cell Disruption by High-Pressure
5 Homogenizer

6 Separation of Cells by Flocculation

7 Batch Sedimentation

8 Cell Separation by Batch Filtration

Extraction of Protein by Aqueous

9 Two-phase Extraction
Protein Concentration by Salting Out
10 Method
Exp No: 1 Date:
ESTIMATION OF PROTEIN BY LOWRY’S METHOD
AIM:

To estimate the amount of protein present in the given unknown solution.

PRINCIPLE:

The Lowry protein assay is a biochemical test that is used to determine the total amount of
protein in the given solution. The total protein concentration is exhibited by the change in colour
of the sample in proportion to protein concentration. The Lowry protein assay uses copper, which
bonds with the peptide bonds in proteins under alkaline conditions. This forms a monovalent
copper ion which can then react with the Folin’s reagent, which in turn can be reduced into a blue-
coloured substance. The protein concentration is calculated by calculating the reactivity of the
peptide nitrogen with the Copper ions under alkaline conditions. Monovalent copper ions undergo
a reduction reaction with Folinciocalteau phosphomolybdic phosphotungstic acid to
Heteropolymolybdenum blue by copper-catalyzed oxidation of aromatic acids. Thus, the
concentration of protein can be determined. However, the method is highly sensitive to the pH of
the assay solution which should be strictly maintained at 10-10.5. But if a very small volume of
sample is used then pH will have the least effect. Some other substance that affects the Lowry
procedure are buffers, sugars, nucleic acids, zwitter ions, lipids and Sulphydryl reagents. Alkaline
CuSo4 catalyzes the oxidation of aromatic amino acids with subsequent reduction of sodium
potassium molybdate tungstate (Folin’s reagent) giving a purple colour complex. The
concentration of reduced Folin’s reagent is measured by absorbance at 600nm.
MATERIALS REQUIRED:
Stock Solution – Bovine Serum Albumin of 1 mg /ml is prepared by using distilled water.
Working Stock – A standard of concentration 0.1 mg/ml is prepared from the above stock
solution.

Folin-phenol Reagent - Folin’s Phenol Reagent is mixed with distilled water in the ratio 1:2.

Reagent A - 2% sodium carbonate in 0.1 N sodium hydroxide.

Reagent B – 1% sodium potassium tartrate in H20

Reagent C – 0.5% copper sulphate in H20

Reagent I – 48ml of reagent A + 1ml of reagent B + 1ml of reagent C

Reagent II- 1-part Folin- phenol reagent:1 part water.

PROCEDURE:

1. The working standard of 0.2 -1ml is pipetted out into a clean test tube and labelled as S1-
S5.
2. The test solution of 0.2ml is taken into a new test tube and labelled as T1. The volume is
made up of 1 ml with distilled water.
3. 1 ml of distilled water serves as blank.
4. To all the test tubes, 4.5ml of alkaline CUSO4 reagent is added and incubated at room
temperature for 10 minutes.
5. 0.5ml of Folin’s phenol reagent is added to all the test tubes from S1 to S5, blank and test
solution.
6. The contents are mixed well using a vortex and checked for the development of blue colour.
7. The intensity of the colour is measured by spectrophotometry at an absorbance of 660 nm
after 15 minutes.
8. Plot the standard graph and calculate the concentration of the given protein.
9. Calculate the concentration of protein in the unknown solution from the standard graph.
TABULATION:

Conc. Vol. of Vol. of

S.No Vol. of Vol. of Incubation OD
of Distilled Reagent
BSA reagent I for at
BSA Water II
(ml) (ml) 30 min 660 nm
(mg/ml) (ml) (ml)

1 B 1.0 4 0.5

2 0.2 0.8 4 0.5

Incubation
3 0.4 0.6 4 10 min 0.5

4 0.6 0.4 4 0.5

5 0.8 0.2 4 0.5

6 1.0 - 4 0.5

Test
7
sample
Exp No: 2 Date:

ESTIMATION OF PROTEINS BY BRADFORD METHOD

AIM: To determine the concentration of protein present in the given samples using
Bradford Assay.

PRINCIPLE:

Bradford assay, first documented by Marion Bradford in 1976, is a fast and fairly
accurate method to estimate protein in a given sample. The Bradford method is a Dye-
based assay and is based on the ability of Coomassie blue to bind protein causing the dye
to shift from a red colour to a blue colour. Coomassie G-250 dye is used as a colourimetric
reagent for the detection and quantification of total protein. In the acidic environment of
the reagent, the protein binds to the Coomassie dye. This results in a spectral shift from
the reddish/brown form of the dye (absorbance maximum at 465 nm) to the blue form of
the dye (absorbance maximum at 610 nm). The difference between the two forms of the
dye is greatest at 595 nm, so that is the optimal wavelength to measure the blue colour
from the Coomassie dye-protein complex. It is thought that both hydrophobic and ionic
forces stabilize the anionic form of the dye, causing a visible colour change. This protein
assay system has a linear working range of 20-200 μg/ml soluble proteins. However, the
presence of detergents in protein samples can cause interference and reduce assay
sensitivity.

MATERIALS REQUIRED:
Test tubes Graduated cylinder
Weight Balance UV spectrophotometer
Bradford reagent
 Dissolve 100 mg of Coomassie-Brilliant blue G250 in 50 ml of 95% EtOH.
Add 100 ml of 85% phosphoric acid and make 600 ml with Distilled H2O.
Filter the solution and add 100 ml of glycerol, then make up to 1000 ml.
The solution can be used 24 h later.
BSA Standard – 0.1 mg/ ml
PROCEDURE:
1. Take clean and dry test tubes labelled and pipette out 0 to 1.0 ml of BSA
working standard and make up the final volume to 1 ml with distilled water.
2. The test tube with 1 ml of distilled water serves as a blank.
3. Add 5.0 ml of Coomassie brilliant blue to each tube and mix by the vortex.
4. Wait for 10-20 minutes and read each of the standards and each of the samples
at 595nm.
5. Plot the standard graph.
6. Estimate the amount of protein present in the given sample from the standard
graph.
TABULATION:

Vol. of Vol of
OD at
S.N Vol of BSA Conc of BSA Distilled Bradford
595
O (ml) (mg/ml) water Reagent
nm
(ml) (ml)

1
0.
2 0.2 5
8
0.
2 0.4 5
6
0.
3 0.6 5
4
0.
4 0.8 5
2
5 1 0 5
0.
Test 0.5 5
5
Exp No: 3 Date:

CELL DISRUPTION BY SONICATION

AIM:

To determine the optimal sonication time corresponding to the maximum protein release
after cell disruption and find the rate constant.

PRINCIPLE:

The sonication process uses ultrasonic sound waves. During the process, there is a
production of thousands of microscopic vacuum bubbles in the solution due to applied pressure.
The formed bubbles collapse into the solution during the process of cavitation. The collapsing of
bubbles takes place in the cavitation field leading to the generation of enormous energy as there is
a production of waves. This results in the disruption of the molecular interactions between the
molecules of water. As there is a reduction in the molecular interactions, the particles start to
separate and allow the mixing process to take place. There is a release of energy from the sound
waves that result in friction in the solution. Ice cubes are used during and after the sonication
process to prevent the sample from heating up. In the ultra-sonication process, cavitation leads to
dispersion, homogenization, disintegration, emulsions, extraction, and sonochemical effects of the
liquids. High-power ultrasound is introduced to the liquid which creates regions of high pressure
(known as compression) and low pressure (known as rarefaction). The creation of these regions is
dependent on the rate of frequency at which the ultrasound is applied. When low pressure is applied
to the liquid, high-intensity ultrasonic waves are produced, creating small vacuum bubbles in the
liquid. As the bubbles reach their saturation level, they collapse and this happens in the high-
pressure cycle. This process is termed cavitation. During cavitation, the bubbles in the liquid can
jet up to 280 m/s velocities.

The below figure explains how the sound wave propagates in the liquid resulting in the
formation of bubbles and their collapse.
 Sonication is the act of applying sound energy to agitate particles in a sample, for various
purposes such as the extraction of multiple compounds from plants, microalgae, and
seaweeds. Ultrasonic frequencies (>20 kHz) are usually used, leading to the process also
being known as ultrasonication or ultra-sonication. Sonication works principally by
converting high-energy sound waves to mechanical energy. The resulting vibrations range
from 18Khz-25Khz. These high-energy waves disrupt intermolecular forces, specifically of
the membrane structures of a cell (sonoporation) hence forcing the cell to release its
intracellular products into the extracellular solution from which further downstream
processing is carried out to obtain the concentrated and purified version of the crude product.
Disruption of the cell membranes occurs through a process called cavitation which involves
the generation of high-pressure minuscule air bubbles. The air bubbles enter gaps in the lipid
membrane and cause a change in the structure of the membrane. Prolonged sonication
ultimately leads to membrane disruption. During the process of sonication, immense heat is
generated from the transfer of energy in the form of rapid consecutive compressions and
rarefaction. To maintain the temperatures of the sample and prevent damage to the cells in
the sample, a coolant of some sort is provided (Liquid coolants, Ice crystals, etc)

Where P = protein content remaining in associated cells

t = time
K is a release constant dependent on the system.
Integrating from P = Pm (maximum possible protein release at time zero) to P = Pt at time
‘t’ gives

As protein (Pr) released from the cells is given by Pr =Pm-Pt, the following equation for cell
breakage is obtained

ln Pm/Pm – Pr = Kt
K = ln (1-R)-1/t,
Where R- Fractional release of protein (The protein release at a specific point divided by the
maximum release attainable)
The constant (K) is independent of cell concentration up to high levels approximately
proportional to the acoustic power above the threshold necessary for cavitation.

MATERIALS REQUIRED:

• Sonicator
• Bacterial sample
• 2ml Eppendorf
• 5ml Eppendorf
• Centrifuge
• Stop clock

PROCEDURE:

1. Take 15 ml of bacterial culture centrifuge tube and centrifuge at 10000 rpm for 5 minutes.
2. Discard the supernatant and resuspend the pellet in 10 ml of distilled water.
3. Set the controller power to 50% and frequency of 25 kHz.
4. Sonicate the sample by varying energy from 100 Joules to 600 Joules of energy.
 5. After 100 Joules of Energy, transfer 1 ml of the sample from the centrifuge to the 1.5 ml
Eppendorf tubes.
6. Centrifuge all the Eppendorf tubes at 10000 rpm for 5 minutes.
7. Take 0.2 ml of supernatant and determine the amount of protein in the sample by
Bradford’s method.
8. Plot the graph between Concentration (mg/ml) vs Energy in joules.

TABULATION:

Concentration
Sonication of released
S.No OD at 595nm ln(P)
Energy protein
(mg/ml) {P}
1 100
2 200
3 300
4 400
5 500
6 600
Exp No: 4 Date:

CELL DISRUPTION BY ENZYMATIC METHOD

AIM:

To study cell disruption by enzymatic method and to measure the protein content.

PRINCIPLE:

Digestion of the cell wall is achieved by the addition of lytic enzymes to a cell suspension.
Enzymes are highly selective, gentle and effective. Lysozyme is widely used to lyses bacterial
cells. The enzyme hydrolyses α-1,4 glycosidic bond in the mucopeptide moiety of the bacterial
cell wall of gram-positive bacteria. The final rupture of the cell often depends on the osmotic
pressure of the suspending medium. In the case of gram-negative bacteria like Escherichia coli
pretreatment with a detergent such as Triton X-100 or the addition of EDTA is necessary. EDTA
is used to destabilize the outer membrane thereby making the peptidoglycan layer accessible to
lysozyme. Yeast cell lysis requires a mixture of different enzymes such as glucanase, protease,
mannanase or chitinase. Sequential disruption of microbial cells for selective product release
involves the use of lytic enzymes based on the type of microbial cells and process conditions.

MATERIALS REQUIRED:

● E.coli culture
● Eppendorf tubes
● Lysis buffer (NaCl, Tris EDTA & Lysozyme (1 mg/ml))
● Alkaline CuSO4
● Folin’s reagent

PROCEDURE:

1. Take 1.5 ml of grown bacterial culture in seven Eppendorf tubes

2. Centrifuge the samples at 10000 rpm for 10 minutes at 4°C
 3. Discard the supernatant and suspend the cell pellet in 1 ml of PBS buffer
4. Add 200 μl of lysis buffer to all the tubes
5. Incubate the tubes at various time intervals (i.e., 10,20,30,40,50 and 60 minutes)
6. Centrifuge the Eppendorf tubes at 10,000 rpm for 5 minutes at 4°C
7. Take the required volume of supernatant and estimate the amount of protein present by
using Bradford’s assay. Plot the column graph between time vs. protein concentration

TABULATION:

Protein
S.No. Time (mins) O.D at 595 nm
Concentration(mg/ml)

1 Control (Untreated)

2 10

3 20

4 30

5 40

6 50

7 60
Exp No: 5 Date:

CELL DISRUPTION BY HIGH-PRESSURE HOMOGENISER

AIM:

To disrupt the cells by using a high-pressure homogenizer at different pressures and to

measure the amount of protein released and rate constant (K).

PRINCIPLE:

A high-pressure homogenizer consists of a high-pressure positive displacement pump

coupled to an adjustable discharge valve with a restricted orifice. The cell suspension is pumped
through the homogenizing valve at 200-1000 atm atmospheric pressure depending on the type of
microorganism and concentration of the cell suspension. The disrupted cell suspension is cooled
as it exits the valve to minimize the thermal denaturation of sensitive products. Cell disruption
occurs due to various stresses developed in the fluid. The primary mechanism seems to be the
stress developed due to the impingement of high-velocity jet-suspended cells on the stationary
surface. The homogenizer including normal and shear stresses generates other stresses. The normal
stress is generated as the fluid passes through the narrow channel of the orifice and the shear stress
as the pressure rapidly reduces to almost atmospheric pressure as the cell suspension passes out of
the orifice. Different parameters influence the degree of cell disruption and rate of release of the
product. These include the nature of the microorganism, its size, cell wall composition and
thickness, concentration of microbial cells, product location within the cell, type of homogenizer,
operating pressure, temperature, and number of passes of the cell suspension through the
homogenizer.
Cell disruption in homogenizer may also be described by first-order kinetic expression as
given by,

ln(Pm/Pm-Pr) = KN Pa

ln (1-R) = -KN Pa

Where N=number of passes through the valve

K is the first order rate constant depending on the

operating pressure

a= 1(Range is 0.9 – 2.9)

Where R- Fractional release of protein (The protein release at a specific point divided by the
maximum release attainable)

PROCEDURE:
1. Set initial pressure as 100 bar
2. Take 500 ml of the given culture sample
3. Homogenize the culture sample for different pressure (100- 500 bar)
4. Repeat the homogenization for two cycles
5. After homogenization, centrifuge the samples at 10000 rpm for 10 minutes at 4°C.
6. Estimate the protein concentration in the supernatant by using Bradford’s method.
7. Plot the column graph between pressure(bar) vs. concentration of protein (mg/ml)
8. Estimate the rate constant (K).
TABULATION:

Conc. of
O.D at
No. of released
S.No. Pressure(bar) 595
passes protein R= Pr/Pm ln (1-R)
nm K
(mg/ml)
Control
1 - - -
(Untreated) -

1
2 100
2

1
3 200
2

1
4 300
2

1
5 400
2

1
6 500
2
Exp No: 6 Date:
FLOCCULATION

AIM:

To clarify the given solution containing cell debris by using different flocculants and to
determine the Debye radius of each flocculant.

PRINCIPLE:

Flocculation is a process where colloids come out of suspension in the form of floc or
flakes. The action differs from precipitation in that, before flocculation, colloids are merely
suspended in a liquid and not dissolved in a solution. In the flocculated system, there is no
formation of a cake since all the flocs are in the suspension. Particles finer than 0.1 µm in water
remain continuously in motion due to electrostatic charge often negative which causes them to
repel each other. Once their electrostatic charge is neutralized by the use of a coagulant chemical,
the finer particles start to collide and agglomerate under the influence of Van der Waals's forces.
These larger and heavier particles are called flocs.

Flocculants, or flocculating agents are chemicals that promote flocculation by causing

colloids and other suspended particles in liquids to aggregate, forming a floc. Flocculants are used
in water treatment processes to improve the sedimentation or filterability of small particles. Many
flocculants are multivalent cations such as aluminium, iron, calcium or magnesium. These
positively charged molecules interact with negatively charged particles and molecules to reduce
the barriers to aggregation. In addition, many of these chemicals, under appropriate pH and other
conditions such as temperature and salinity, react with water to form insoluble hydroxides which,
upon precipitating, link together to form long chains or meshes, physically trapping small particles
into the larger floc.

MATERIALS REQUIRED:

1. Cell culture
2. Aluminum sulphate
3. Calcium chloride
4. Potassium chloride
5. Sodium chloride

PROCEDURE:

1. 5 ml of cell debris solution was taken in 4 different test tubes.

2. 2 ml of flocculants were added to the corresponding test tubes and left for 30 minutes
incubation.
3. After settling of microflocs, the supernatant is collected.
4. Absorbance was measured at 600nm in spectrophotometer.

TABULATION:

S.No Flocculants Volume of O.D at 600 nm Debye radius(m)

flocculants (ml) (rDR)

Aluminum sulphae

Calcium chloride

Potassium chloride

Sodium chloride
Exp No: 7 Date:

BATCH SEDIMENTATION

AIM:

To study the settling velocity of solid particles under batch sedimentation and to find the
area of the continuous thickener.

PRINCIPLE:

There are several stages in the settling of flocculated suspension and different zones are
formed as sedimentation proceeds. Usually, the concentration of the solids is high enough, that
sedimentation of individual particles or flocs is hindered by other solids to such an extent that all
solids at a given level settle at a common velocity. At first, the solid is uniformly distributed in the
liquid, the total depth of the suspension is Z0. After a short time, the solids settle to give a zone of
clear liquid.

Design of Thickeners

The thickening process takes place in a settling tank with a long enough solids retention
time. For example, in secondary clarifiers of activated sludge systems both clarification and
thickening operations are carried out. The thickening of the sludge is a concern to the operator who
desires a high underflow solids concentration. So it is the general practice to design these processes
for both thickening and clarification performance. Similar to the digesters, there are two design
approaches in thickeners too:

1. Design based on experience

2. Design based on Laboratory data

Design based on Laboratory data

It is the best technique if the Laboratory data is available. A typical test is done by using
a 1000mL-graduated cylinder. Sludge (Calcium carbonate) is mixed homogenously and let to settle
in the cylinder. In seconds an interface separating the solids and the clear water on top is formed
with a certain settling velocity. This velocity of the interface is measured with respect to time.
Interface height is plotted against time and the zone settling velocity (ZSV) is calculated from the
initial slope of the graph. The graph is given in Figure 1. The velocity with which solids settle out
will depend on the concentration of solids. Right after time zero, two interfaces are moving towards
each other. One from the bottom up due to the building up layers of sludge from the bottom, the
other interface is moving down from top to bottom, this is the blanket of settling sludge, settling
velocity, v. At time t2, these will meet and settling will slow down. Then the settling will cease
over time and compaction begins [3]. Figure 2 shows the illustration of interfaces during the
settling test.
 (i) Area of the thickener = FC0/(VL/ (1/CL-1/Cu)) m2
(ii) VL= (Zi – ZL)/θL
(iii) Ci = C0Z0/Zi
Where,

F = Flow rate(m3)
C0 = Initial concentration of the slurry (kg/m3) at time 0
Cu = Underflow concentration (kg/m3)
VL = Velocity of settling (m/s)

PROCEDURE:
1) A slurry of 5% concentration of calcium carbonate was prepared in a 1-lit graduated
measuring cylinder.
2) The slurry was stirred well using a stirrer and the level of liquid at time t equal to zero was
noted down
3) The level of interface between the clear liquid and slurry of original concentration at
various time intervals was noted
4) The values of level ht and time t were tabulated
5) A graph was plotted between height vs. time t.
TABULATION:

Table 1

Time Height of interface

S. No
(sec) cm
Table 2

CL = C0Z0/Zi VL/(1/CL-1/CU)
Zi ZL (m) θL (s) VL = Zi-ZL/θL (m/s)
(kg/m3) (kg/m2s)
(m)
Exp No: 8 Date:
BATCH FILTRATION
AIM:
To filter the given slurry and to determine the specific cake resistance α and medium
resistance Rm.

PRINCIPLE:

Filtration is the conventional unit operation aimed at the separation of a particular matter.
Filtration is defined as the separation of solid in a slurry consisting of the solid and fluid
bypassing the slurry through a septum called filter medium. The filter medium allows the fluid
to pass through and retains the solid. The separated solid called the filter cake forms a bed of
particles on the filter medium. The thickness of the cake increases from an initial value of zero
to the final thickness at the end of filtration.

The rate of filtration is defined as the volume of filtrate collected per unit time per unit
area of the filter medium. The resistance of the medium is constant and independent of the cake.

MATERIALS REQUIRED:

Calcium carbonate solution (10%), filter paper, funnel, measuring cylinder

PROCEDURE:

1. 50 ml of 10% CaCo3 was taken and filtered through the filter paper by using a funnel
2. The volume of filtrate collected was noted down in the measuring cylinder for every
minute until the end of the filtration
3. The surface area of the funnel was calculated
4. The graph was plotted between At/V Vs V/A and the slope and intercept were calculated
from the graph
5. From the values of slope and intercept the specific cake resistance (α) and medium
resistance (Rm) were calculated.
TABULATION:

Time (sec) Volume of the filtrate (ml) At/V (sec/cm) V/A (cm)

CALCULATION:

(At/ V) = K (V/A) + B

Slope = µαρ / ∆p; B = µ Rm / ∆p

Exp No: 09 Date:

EXTRACTION OF PROTEIN BY AQUEOUS TWO-PHASE METHOD

AIM:
To measure the amount of protein present in the given sample using Aqueous Two-phase
extraction (ATPE).

PRINCIPLE:
Biomolecules and cellular particles can be separated and purified by partition between two
immiscible aqueous polymer phases. By this method, macromolecules and particles are separated
according to their surface properties. The separation can be based on size, electric charge,
hydrophobicity or specific recognition. The method can be made highly selective; it preserves
biological applications with good economy.
Aqueous two-phase systems (ATPs) are generally composed of a water solution of
structurally distinct hydrophilic polymers (e.g. polyethene glycol, dextran) or one polymer and
certain salts (e.g. ammonium sulphate, potassium phosphate). Success with aqueous two-phase
systems depends on the ability to manipulate phase composition to obtain appropriate partition
coefficients (K) and selectivity for the material of interest.
There are several ways to manipulate system composition to give phases with appreciably different
physical properties and the three below are relatively important:
a) Choice of polymers, polymer concentration, polymer molecular weight
b) Choice of salts(s) and salt concentration and
c) Chemical modification of one of the polymers by attaching a ligand for which receptors
exist on the material of interest.
In the last case, the resulting procedure is called affinity partitioning.

Aqueous solutions of most hydrophilic polymers such as starch, gelatin or agar show
incompatibility in binary mixtures and form two immiscible aqueous phases, each containing
primarily only one of the two phases forming polymers and a high proportion of water. At low
concentrations of the polymers, homogenous solutions (single phase) are obtained but at discrete
concentration ratios measurably by the cloud point method, phase separation occurs. The mutual
insolubility of the two hydrophilic polymers may be attributed to the molecular form of each
polymer, which results in mutual repulsion.

Polymer: Salt Two Phase systems

Many polymers form two-phase liquid-liquid systems when combined with suitable salts
(e.g. Phosphates and sulphates). Because of their low costs and short settling time, polyethene
glycol/ salt systems have been found used for the technical extraction of enzymes.
Partition:
The partition of a solute (e.g. a protein) between the phases is described by a partition
coefficient, K. defined as the ratio between the concentrations of solute in the upper and lower
phases.
Kpart = O.D or Protein concentration of top phase Ctop / O.D or protein of bottom phase
(Cbottom)
V T x O.D top
Yield = --------------------; VT - Volume of top phase
V B x O.D bottom VB - Volume of bottom phase

Polymer Polymer/ Low Molecular Weight Substance

Polymer-polymer system Dextran

PVA
PEG PVP
Polypropylene glycol Ficoll
Dextran, PVA
PVA Polyvinylpyrrolidone
Polyvinyl pyrrolidone Dextran
Methylcellulose
Polymer – Low molecular weight substance
systems

Potassium phosphate
PEG
Polyvinyl pyrrolidone Potassium phosphate

Polypropylene glycol Glucose

Dextran Propyl alcohol
Sodium dextran sulphate
Sodium chloride
Polyelectrolyte: Non-ionic + Ionic systems PEG and Sodium chloride
Sodium dextran sulphate
PVA and Sodium chloride
Sodium carboxymethylcellulose
PEG
PVA

PROCEDURE:
1. 45% (w/w) of Sodium phosphate (i.e. 45 g of salt in 65 g of water and 50% of PEG 6000
– 50 g of PEG 6000 in 50 g of water) was prepared
2. The protein solution was prepared (1 g BSA/ 10 ml of water)
3. To the protein solution 5 ml of salt solution was added
4. Then drop by drop 5 ml of PEG 6000 was added
5. The mixture was allowed to stand for some time to obtain the two phases.
6. The contents were thoroughly mixed.
7. It was allowed to settle for 20 minutes and the two phases were separated by careful
pipetting and the volumes were noted down.
8. The partition coefficient and Yield were calculated using the given formulae.
Exp No: 10 Date:

PROTEIN PURIFICATION BY SALTING OUT METHOD

AIM
To find % protein recovery from precipitation of proteins from the given solution by
adding ammonium sulfate.

INTRODUCTION
Ammonium sulphate precipitation is a method of protein purification by altering the
solubility of protein. It is a specific case of a more general technique known as salting out.
Ammonium sulfate is commonly used as its solubility is so high that salt solutions with high ionic
strength are allowed.
The solubility of protein varies according to the salt concentration. Two distinct effects
are observed: at low salt concentrations, the solubility of the protein increases with increasing salt
concentration (i.e. increasing ionic strength), an effect termed salting in. As the salt concentration
(ionic strength) is increased further, the solubility of the protein begins to decrease. At sufficiently
high ionic strength, the protein will be almost completely precipitated from the solution (salting
out).
Since proteins differ markedly in their solubility at ionic strength, salting out is a very
useful procedure to assist in the purification of a given protein. The commonly used salt is
ammonium sulfate, as it is very water soluble and has no adverse effects on enzyme activity. It is
generally used as a saturated aqueous solution which is diluted to the required concentration,
expressed as a percentage concentration of the saturated solution (a 100 % solution)

LIST OF REAGENTS AND INSTRUMENTS:

A. Equipment
● Test tubes
● Graduated cylinder
● Pipettes
● Balance
● Centrifuge
Reagents
● Prepare a known concentration protein solution(BSA – 2 mg / 10 ml)
● Prepare various percentages of Saturated (NH4)2SO4 solution according to the standard
chart
PROCEDURE:

1. Various conc. of saturated ammonium sulphate solution. (20%, 40%, 60%, 80%, 90%,
100%) was prepared
2. 10ml of BSA solution or disrupted cell culture was taken into a test tube.
3. The specified amount of ammonium sulfate slowly added to the test tube
4. Protein precipitation is not instantaneous; it may require 20--30 minutes to equilibrate.
5. The solution was centrifuged for 10,000 rpm for 10 mins
6. The precipitate was collected carefully discarding as much supernatant as possible
7. To the precipitate 100 µl of PBS buffer was added to dissolve the pellet
8. The protein concentration measured by Lowry’s method
9. Protein Recovery is calculated by using the formula

Final protein content

% Protein Recovery = ------------------------- x 100
Initial protein content

TABULATION:
Table 1
S.No Ammonium sulphate (%) g/L
1 20 106
2 40 226
3 60 361
4 80 516
5 90 603
6 100 697
Table 2
S.No Ammonium O.D at 540 nm Protein concentration Protein
sulphate (%) (mg/ml) Recovery (%)