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

Isoelectric Focusing

2D Gel Electrophoresis

Ultracentrifugation

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