Transcript Chapter 3 (part 2)
Chapter 3 (part 2) Protein purification and Analysis
Why purify proteins?
• Pure proteins are required to study enzyme function • Pure proteins are required for structural analysis (x-ray crystallography, NMR spectroscopy) • Pure proteins are required to obtain amino acid sequence
Steps in protein purification • Develop assay • Choose source of protein • Prepare tissue extract – cell disruption – subcellular fractionation • Protein fractionation (several steps) • Determination of purity
Differential Centrifugation transfer supernatant transfer supernatant transfer supernatant
1000 g 10,000 g 100,000 g
tissue homogenate Pellet unbroken cells nuclei chloroplast Pellet mitochondria Pellet microsomal Fraction (ER, golgi, lysosomes, peroxisomes) Super.
Cytosol, Soluble enzymes
Chromatography
Gel Permeation Chromatography
Ion-exchange Chromatography + + + + + + + + + + + + + - - - - + + + + + + + + + + + + + + low salt buffer + + + + + - - + + + + + + + + + + + + + + + + + + + + + high salt buffer Cl + Cl + + + + + + + + + + + Cl Cl + Cl + + + + + + + + + + - - - - - - -
Affinity Chromatography Add excess ligand
SDS poly acrylamide electrophoresis (PAGE) SDS = H 3 C-(CH 2 ) 10 -CH 2 -OSO 3 SDS – denatures protein coats w/ negative charge - Used to determine protein MW And purity of protein prep
pH 9 Isoelectric Focusing pH 3 + +
large small 2-D Electrophoresis Decreasing pH SDS-PAGE + Decreasing pH
Amino Acid Analysis 1) Acid hydrolyze protein 2) Treat with phenylisothiocyanate (PICT) N C S + H 3 N H C O C O R N S C HN C C R H O 3) Separate derivatized AA’s by HPLC
1) Protein Sequencing (Edman Degradation) H O N C S H 3 N C C NH H C O C R R X 2) N S C HN H C O C H C O C X NH 3) R R Trifluoroacetic acid N S C C HN C R H + 2HN H C R O C X O Can sequence in 30 to 60 AA’s from N-terminus
Generate Proteolytic Fragments Endopeptidases •Typsin •Chymotrypsin Chemical Cleavages cleaves at COOH end of Lys and Arg cleaves at COOH end of Phe, Tyr, Trp •Cyanogen Bromide cleaves at COOH end of Met Generate overlapping fragments Sequence individual fragments and piece together sequence
Peptide mapping exercise Met-Ala-Arg- Gly-Glu-Tyr-Met-Cys-Lys-Phe-Ala-Glu-Gln-Asp Trypsin Met-Ala-Arg Phe-Ala-Glu-Gln-Asp Gly-Glu-Tyr-Met-Cys-Lys Chymotrysin Met-Ala-Arg- Gly-Glu-Tyr Met-Cys-Lys –Phe Ala-Glu-Gln-Asp CNBr Met Ala-Arg-Gly-Glu-Tyr-Met Cys-Lys-Phe-Ala-Glu-Gln-Asp
Proteomic Analysis
M atrix A ssisted L aser D esorption I onization T ime o f F light (MALDI-TOF)