Purification of Protiens: Mohan CC M.SC, Biochemistry 1 Semester

PURIFICATION OF PROTIENS
Mohan CC
M.Sc , Biochemistry
1st Semester
Guide
Dr. Naveen Y.P
PROTEINS INTRODUCTION :
 Proteins are the bio molecules made up of amino

acids , and each amino acids are bonded to each other
by peptide bond.
 The peptide bond is formed by the bonding

between the amide group and the carboxil group of
the amino acids.
WHY DO WE NEED PURIFIED
PROTEIN ?
 By analyzing pure proteins, we can determine amino acid
sequences and can investigate protein’s biochemical functions.
 Even for the process of crystalization of proteins we need pure

proteins
 Through this process we can observe the structures of proteins,

specially tertiary structure , which is the functional unit of the
protein.
PURIFIED PROTEIN ;
 It is the targeted protein . we need assay to identify the specific
protein molecule or the targeted protein molecule.
IDENTIFICATION OF TARGETED
PROTEIN :
BLOTTING :
 It is one of the blotting methods which are used in the

separation of the protein molecules.
PROCESS OF PURIFICATION :
 HOMOGENISATION.
 CENTRIFUGATION.
 PRECIPITATION.
 DIALYSIS.
 CHROMATOGRAPHY
 ELECTROPHOROSIS.
 MASS SPECTROSCOPY
HOMOGENISATION :
1] SONICATION.
 It is the process of rupturing of cells through the sound waves.

 When the sonicator is switched on , the sound waves
hit the solution containing the cells leading in their rupture.
CHEMICAL CLEVAGE :
 Cells are destroyed using the ethanol or aceticacid.
 The cell membranes can also be disrupted using lysozomes.
PHYSICAL METHOD :
 Using blenders, magnetic blenders, and homogenizers.
CENTRIFUGE :
 It is the process where the particles or the molecules are

separated due to the centrifugal force.
NOTE :
PROTEINS CAN BE SEPARATED

ACCORDING TO THEIR ;
 Solubility
 Shape
 Charges
 Specific binding affinity.
SALTING IN AND OUT :
{ PRECIPITATION REACTION }
DIALYSIS :
 Used to separate proteins on their size.
 Semi permiable membrane is used for separation .
 Cellulose membrane is used as semi permiable membrane

.
 But, could not distinguish between proteins efficiently.

CHROMATOGRAPHY :
 It is the most distinguished form to separate from the size,
shape and from charges of the proteins.
1] GEL FILTRATION CHROMATOGRAPHY . (

BY SIZE )
 Consists of beads.
 Small molecules passes through the beads and also from the
pores of the beads.
 Large molecules simply pass between the space of the beads.
 Small molecules take time whereas, large molecules moves

rapidly.
2] ION EXCHANGE CHROMATOGRAPHY :

( BY CHARGE )
 Proteins have different charges, also varies due to pH of the
surroundings.
 Positive protein molecules binds to the negative beads , that is
“ Carboxylate ” group.
 Whereas , negative charge binds to positive beads , that is

“ Diethylaminoethyl ” group.
 The flow depends on chromatogram and the protein charged .
 The left over protein can be removed by the running of high

concentrated salt solution through it.
 The Na+ ions reacts with the negative / Cl- ions react with the
positive beads and removes the leftover protein in the
chromatographic chamber.
 Correct chromatogram should be used for the correct results of

the protein sample.
3] AFFINITY CHROMATOGRAPHY :
IT’S A POWERFUL AND MOST APPLICABLE TECHNIQUE .
TAKES ADVANTAGE OF THE HIGH AFFINITY OF MANY PROTEINS FOR

SPECIFIC AMINO ACID GROUPS.
EX:- “ Nipolysterine” , widely used for His-tag protein.
 His-tag has widely affinity towards nipolysterine and attaches to

chromatographic chamber, while others don’t .
 Transcription factors can also be used .
 The specific proteins attaches to the factor. Whereas, others

don’t .
 Then they are washed by excess concentration of salt solution.
Conclusion :-
Chromatography depends on ;
 Attaching , Loading sample, Elucidating .

HPLC ( HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY ):-
 HPLC is the technique used to enhance the resolving power of
all the chromatographic technique.
 Samples are loaded to the chamber with high pressure, in

stationary phase.
 The object leaves the column at different point
 Thus, the detector gains different peak signals . each of these

peaks represents different kind of compound.
 After comparing the result, we know the sample containing

material.
 The high resolution as well as rapid separation helps in the net

result.
QUALITATIVE AND QUANTITATIVE ANALYSIS :
ELECTROPHOROSIS :-
SDS-PAGE :-
( SODIUM DODICYL SULPHATE - POLY
ACRYLAMIDE GEL ELECTROPHOROSIS )
 It’s a method of separation of proteins .
 DIG ( digoxigenin ) is used as primary antibody.
 Secondary anti bodies are used
 Then the protein of low molecular weight flows fast in

comparison to the high weight molecules .
 SDS acts as a detergent .
 The initial fractions will display dozens to hundreds of protein .

 As the purification progress, the number of bands will deminish
and prominance of one of the band increases .
 The band corresponds to the protein of interest .

 MASS SPECTROSCOPY :-
 It can determine the mass of the required protein sufficiently and

exceptionally .
 This process is called matrix assisted laser desorption ionisation

( MALDI ) .
 Using this we can learn even more about the protein of interest.

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PURIFICATION OF PROTIENS

 Proteins are the bio molecules made up of amino

 The peptide bond is formed by the bonding

 Even for the process of crystalization of proteins we need pure

 Through this process we can observe the structures of proteins,

 It is one of the blotting methods which are used in the

 It is the process of rupturing of cells through the sound waves.

 Cells are destroyed using the ethanol or aceticacid.

 The cell membranes can also be disrupted using lysozomes.

 It is the process where the particles or the molecules are

PROTEINS CAN BE SEPARATED

 Semi permiable membrane is used for separation .

 Cellulose membrane is used as semi permiable membrane

 But, could not distinguish between proteins efficiently.

1] GEL FILTRATION CHROMATOGRAPHY . (

 Large molecules simply pass between the space of the beads.

 Small molecules take time whereas, large molecules moves

2] ION EXCHANGE CHROMATOGRAPHY :

 Whereas , negative charge binds to positive beads , that is

 The flow depends on chromatogram and the protein charged .

 The left over protein can be removed by the running of high

 Correct chromatogram should be used for the correct results of

IT’S A POWERFUL AND MOST APPLICABLE TECHNIQUE .

TAKES ADVANTAGE OF THE HIGH AFFINITY OF MANY PROTEINS FOR

 His-tag has widely affinity towards nipolysterine and attaches to

 Transcription factors can also be used .

 The specific proteins attaches to the factor. Whereas, others

 Then they are washed by excess concentration of salt solution.

 Attaching , Loading sample, Elucidating .

 Samples are loaded to the chamber with high pressure, in

 The object leaves the column at different point

 Thus, the detector gains different peak signals . each of these

 After comparing the result, we know the sample containing

 The high resolution as well as rapid separation helps in the net

 DIG ( digoxigenin ) is used as primary antibody.

 Secondary anti bodies are used

 Then the protein of low molecular weight flows fast in

 SDS acts as a detergent .

 The initial fractions will display dozens to hundreds of protein .

 The band corresponds to the protein of interest .

 It can determine the mass of the required protein sufficiently and

 This process is called matrix assisted laser desorption ionisation

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