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EXPLORING PROTEINS AND PROTEOMES GENOMES AND PROTEOMES OBTAINING THE PROTEIN: CELL LYSIS AND SEPARATION PROTEIN PURIFICATION TECHNIQUES Salting out HPLC Dialysis Gel Electrophoresis Isoelectric Focusing 2D Gel Electrophoresis Ultracentrifugation Gel Filtration Chromatography Ion-exchange Chromatography Affinity Chromatography SALTING OUT DIALYSIS GEL FILTRATION CHROMATOGRAPHY ION-EXCHANGE CHROMATOGRAPHY AFFINITY CHROMATOGRAPHY ELISA HPLC GEL ELECTROPHORESIS ISOELECTRIC FOCUSING 2D GEL ELECTROPHORESIS ULTRACENTRIFUGATION STRUCTURE DETERMINATION AMINO ACID SEQUENCING: EDMAN DEGRADATION 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 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 MASS SPECTROMETRY MALDI-TOF MS ELECTROSPRAY IONIZATION X-RAY CRYSTALLOGRAPHY X-RAY DIFFRACTION PATTERN OF MEVALONATE KINASE NMR SPECTROSCOPY
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