Protein purification techniques involve isolating a single protein from a complex mixture in order to characterize its function, structure, and interactions. The main steps include extracting proteins from cells, stabilizing them in solution, separating them using techniques like precipitation, filtration, dialysis, and various types of chromatography, and then verifying the purity of the isolated protein using analytical methods like SDS-PAGE and mass spectrometry. The basis of protein separation includes differences in solubility, binding interactions, size, charge, and other properties.
2. PROTEIN PURIFICATION
To isolate a single type of protein from a complex
mixture
vital for characterization of
• function
• structure
• interactions of the protein of interest.
Starting material is a biological tissue or a microbial
culture
3. PURPOSE
To identify the function of a protein
To identify the structure of a protein
To produce a commercial product
To produce a large quantity of purified proteins like
enzyme etc.
To produce a small quantity of a protein for research
purposes
4. STEPS FOR PROTEIN
PURIFICATION
Extracting pure proteins from cells
Stabilizing the proteins in solution
Separation :
Protein precipitation (salting in and salting out) ,
Filtration , dialysis
Chromatography: ( gel filtration , ion exchange ,
affinity chromatography, hydrophobic interaction
chromatography , isoelectric focusing)
Analytical methods ( HPLC , MS)
Verification ( SDS PAGE , western blotting , ELISA )
5. BASIS OF PROTEIN
SEPARATION
Solubility
Binding interactions
Surface-exposed hydrophobic residues
Charged surface residues
Isoelectric point
Size and shape
6. EXTRACTION
For exraction ,cell lysis is required which is done by:
osmotic lysis
freeze- thaw lysis
detergent lysis
enzymatic lysis
7. STABILIZATION
goal is to maintain biological activity
Threats are:
temperature
proteases
mechanical destruction
SOLUTION:
use additives, protease inhibitors and maintain
temperature
8. PROTEIN PRECIPITATION
Ammonium sulphate precipitation:
purify proteins by altering their solubility
Salting in:
At low salt conc, protein solubility increases by
increasing salt conc
Salting out:
protein solubility decreases by further increase in
salt conc
Dehydration of proteins results in precipitation
9. DIAYSIS
Done to remove ammonium sulphate
Passage of solutes through semipermeable membrane
Pores in dialysis membrane are of certain size
remove ammonium and sulphate ions
not allow passage of proteins
15. HYDROPHOBIC INTERACTION
CHROMATOGRAPHY
Resins used in the column are amphiphiles
The hydrophobic part of the resin attracts hydrophobic
region on the proteins
greater the hydrophobic region on the protein, stronger
the attraction between the gel and protein
25. PROTEIN PRESERVATION
preservation is done by lyophilization
Lyophilization/ freeze drying
• Sample is frozen
• Vacuum for removal of water
• Ice crystals sublimate to water vapour directly
• Containers are sealed