1. E XPLORING P ROTEINS AND P ROTEOMES

2. G ENOMES AND P ROTEOMES

3. O BTAINING THE PROTEIN : CELL LYSIS AND S EPARATION

4. P ROTEIN P URIFICATION T ECHNIQUES Salting out Dialysis Gel Filtration Chromatography Ion-exchange Chromatography Affinity Chromatography HPLC Gel Electrophoresis Isoelectric Focusing 2D Gel Electrophoresis Ultracentrifugation

5. S ALTING O UT

6. D IALYSIS

7. G EL F ILTRATION C HROMATOGRAPHY

8. I ON - EXCHANGE C HROMATOGRAPHY

9. A FFINITY C HROMATOGRAPHY

10. ELISA

11. HPLC

12. G EL E LECTROPHORESIS

13. I SOELECTRIC F OCUSING

14. 2D G EL E LECTROPHORESIS

15. U LTRACENTRIFUGATION

16. S TRUCTURE D ETERMINATION

17. A MINO A CID S EQUENCING : E DMAN D EGRADATION

18. B ROAD C LASSIFICATION OF P ROTEASE Serine Proteases A serine residue acts as a nucleophile in the active site, facilitating the reaction Operates via the catalytic triad His, Ser- Asp Cystein Proteases Cys as the nucleophile and activated by a nearby basic aa Papain is an example Aspartate Proteases Two Asp residues cleaves the peptide bond by activating a water molecule Metalloprotease A metallic Zn or Co is involved in the catalytic mechanism

19. E XAMPLES OF P ROTEASES Trypsin – carboxyl side of Lys and Arg Chymotrypsin – carboxyl side of bulky hydrophobic aa Elastase – carboxyl side of small hydrophobic aa Clostripain – carboxyl side of Arg Thrombin – serine protease with specific cleavage site: Leu-Val-Pro-Arg—Gly-Ser Carboxypeptidase A – amino side of C terminal residues of aromatic or aliphatic aa An exopeptidase

20. M ASS S PECTROMETRY

21. MALDI-TOF MS

22. E LECTROSPRAY I ONIZATION

23. X- RAY C RYSTALLOGRAPHY

24. X- RAY D IFFRACTION P ATTERN OF M EVALONATE K INASE

25. NMR S PECTROSCOPY