PPT protein separation and purification

special study for all master and BSc student
protein isolation,
protein purification
protein separation,
protein analysis
and some extra like end group analysis ( n-terminal, and c-terminal) etc
i'm kaushal kumar sahu msc final year biotechnology..

1. Protein Separation and Purification Guided by Presented by Dr. Prerna Soni Kaushal Kumar Sahu 25 /11/2017 Seth Phoolchand Agrawal College

2. Protein separation and purification Introduction Definition Separation Extracellular Intracellular Purification Differential centrifugation Differential salt precipitation Electrophoresis Different chromatography Conclusion Reference Recent Research  

3. Introduction The purification process may separate the protein and non-protein parts of the mixture, and finally separate the desired protein from all other proteins .. Separation steps usually exploit differences in protein size, physico -chemical properties, binding affinity and biological activity.

4. Purpose include the preparation of commercial products such as enzymes (e.g. lactase ), nutritional proteins (e.g. soy protein isolate), certain biopharmaceuticals (e.g. insulin). research or analytical purposes , including identification , quantification, and studies of the protein's structure , post-translational modifications and function.

5. Separation: - Extracellular:- No need for cell disruption Secreted soluble proteins can be collected in the cell supernatant after centrifugation Membrane-bound proteins might be released from the cell simply using detergents

6. Need cell disruption Detergents lysis Enzymatic lysis Osmotic lysis Ultrasonication Intracellular

7. Purification:- Differential centrifugation :- Force Time sediment 1000g 5min Eu. cell 4,000g 10min Chl,cell debris, nuclei 15,000 20min Bacteria, mito 30,000 30min Small organelle 200,000 1hr Small vesicle 100,000 3-10hrs Ribosome 200,000 10-24hrs Membrane sheets

8. Differential salt precipitation

9. Different chromatography Column Chromatography :- •Most common and best approach to purifying larger amounts of proteins •Achieves highest level of purity and largest amount •Requires low effort •Lowest likelihood to damage the protein product •Standard method for pharmaceutical industry

10. Size-Exclusion (or molecular exclusion ) Chromatography •Molecules are separated according to differences in their size as they pass through a hydrophilic polymer •Polymer beads composed of cross-linked dextran (dextrose) which is highly and uniformly porous (like Swiss cheese) •Large proteins come out first (can’t fit in pores), small proteins come out last (get stuck in the pores)

11. Principle is to separate on basis of charge “adsorption” Highest resolving power Highest loading capacity Widespread applicability Most frequent chromatographic technique for protein purification Ion Exchange chromatography

12. Adsorptive separation in which the molecule to be purified specifically and reversibly binds (adsorbs) to a complementary binding substance (a ligand ) immobilized on an insoluble support (a matrix or resin) •Purification is 1000X or better from a single step (highest of all methods) •Preferred method if possible Affinity chromatography

13. Polyacrylamide Gel Electrophoresis Electrophoresis separates proteins based on their size using electrical current

14. protein are usually soluble in water solution because they have hydrophilic amino acids on their surface that attract water molecules and interact with them. Conclusion

15. Reference:- Bioanalytical techniques, M. L. Srivastava (2008), Narosa Publication House, ISBN 978-81-7319-852-6 Principles of Biochemistry, David L. Nelson And Michael M. Cox (2008), W H FREEMAN And Company, ISBN-10: 0-7167-7108-X Molecular Cell Biology, LODISH And et.al. (2016), W H FREEMAN And Company, ISBN10:1-4641-8339-2