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.

1. Protein Purification techniques

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

10. DIAYSIS

11. CHROMATOGRAPHY
 separating mixtures into components depending upon difference
in
• size
• charge
• mass
• solubility
• Adsorption

12. SIZE EXCLUSION
CHROMATOGRAPHY
 Using porous matrix
 Based on different sizes of proteins

13. ION EXCHANGE
CHROMATOGRAPHY
 Anion exchange resins (+ve) separate negatively
charged compounds
 cation exchange resins (-ve ) separate positively
charged molecules

14. AFFINITY
CHROMATOGRAPHY
 separation technique based upon molecular
conformation
 resins have ligands attached to their surfaces which are
specific for the compounds to be separated

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

16. ISOELECTRIC FOCUSING

17. 2D GEL ELECTROPHORESIS

18. ANALYTICAL TECHNIQUES
 HPLC
 Mass spectrometery

19. HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY

20. MASS SPECTROMTERY

21. VERIFICATION
 SDS-PAGE
 Western blotting
 ELISA

22. SDS-PAGE

23. WESTERN BLOTTING

24. ELISA

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

26. LYPHOLIZATION STEPS

27. APPLICATIONS