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EXPLORING PROTEINS AND PROTEOMES

E XPLORING P ROTEINS AND P ROTEOMES. G ENOMES AND P ROTEOMES

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O BTAINING THE PROTEIN : CELL LYSIS AND S EPARATION

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Page 1: E XPLORING P ROTEINS AND P ROTEOMES. G ENOMES AND P ROTEOMES

EXPLORING PROTEINS AND PROTEOMES

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GENOMES AND PROTEOMES

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OBTAINING THE PROTEIN: CELL LYSIS AND SEPARATION

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PROTEIN PURIFICATION TECHNIQUES Salting out

Dialysis

Gel Filtration Chromatography

Ion-exchange Chromatography

Affinity Chromatography

HPLC

Gel Electrophoresis

Isoelectric Focusing

2D Gel Electrophoresis

Ultracentrifugation

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SALTING OUT

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DIALYSIS

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GEL FILTRATION CHROMATOGRAPHY

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ION-EXCHANGE CHROMATOGRAPHY

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AFFINITY CHROMATOGRAPHY

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ELISA

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HPLC

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GEL ELECTROPHORESIS

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ISOELECTRIC FOCUSING

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2D GEL ELECTROPHORESIS

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ULTRACENTRIFUGATION

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STRUCTURE DETERMINATION

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AMINO ACID SEQUENCING: EDMAN DEGRADATION

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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

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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

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MASS SPECTROMETRY

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MALDI-TOF MS

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ELECTROSPRAY IONIZATION

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X-RAY CRYSTALLOGRAPHY

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X-RAY DIFFRACTION PATTERN OF MEVALONATE KINASE

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NMR SPECTROSCOPY