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Exploring Proteins and Proteomes

Exploring Proteins and Proteomes. Genomes and Proteomes. Obtaining the protein: cell lysis and Separation. Protein Purification Techniques. Salting out Dialysis Gel Filtration Chromatography Ion-exchange Chromatography Affinity Chromatography. HPLC Gel Electrophoresis

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Exploring Proteins and Proteomes

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  1. Exploring Proteins and Proteomes

  2. Genomes and Proteomes

  3. Obtaining the protein: cell lysis and Separation

  4. Protein Purification Techniques • Salting out • Dialysis • Gel Filtration Chromatography • Ion-exchange Chromatography • Affinity Chromatography • HPLC • Gel Electrophoresis • Isoelectric Focusing • 2D Gel Electrophoresis • Ultracentrifugation

  5. Salting Out

  6. Dialysis

  7. Gel Filtration Chromatography

  8. Ion-exchange Chromatography

  9. Affinity Chromatography

  10. ELISA

  11. HPLC

  12. Gel Electrophoresis

  13. Isoelectric Focusing

  14. 2D Gel Electrophoresis

  15. Ultracentrifugation

  16. Structure Determination

  17. Amino Acid Sequencing: Edman Degradation

  18. Broad Classification of Protease • 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. Examples of Proteases • 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. Mass Spectrometry

  21. MALDI-TOF MS

  22. Electrospray Ionization

  23. X-ray Crystallography

  24. X-ray Diffraction Pattern of Mevalonate Kinase

  25. NMR Spectroscopy

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