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.