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Protein purification and Analysis

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.

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Protein purification and Analysis

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  1. Protein purification and Analysis

  2. 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

  3. Steps in protein purification • Develop assay • Choose source of protein • Prepare tissue extract • cell disruption • subcellular fractionation • Protein fractionation (several steps) • Determination of purity

  4. Differential Centrifugation transfer supernatant transfer supernatant transfer supernatant 1000 g 10,000 g 100,000 g Pellet microsomal Fraction (ER, golgi, lysosomes, peroxisomes) Pellet unbroken cells nuclei chloroplast Pellet mitochondria Super. Cytosol, Soluble enzymes tissue homogenate

  5. Chromatography

  6. Gel Permeation Chromatography

  7. + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ Ion-exchange Chromatography low salt buffer high salt buffer - - - - - - - - - Cl- - - - Cl- Cl- Cl- - - - Cl- Cl- - - - - - - - - - - - -

  8. Affinity Chromatography Add excess ligand

  9. - - - - - - - - - - - - - - - - SDS poly acrylamide electrophoresis (PAGE) SDS = H3C-(CH2)11-CH2-OSO3- SDS – denatures protein coats w/ negative charge Used to determine protein MW And purity of protein prep

  10. Isoelectric Focusing - - pH 9 Decreasing pH Decreasing pH pH 3 + +

  11. - Decreasing pH + 2-D Electrophoresis Decreasing pH - SDS-PAGE Decreasing MW + Decreasing pH

  12. Amino Acid Analysis • Acid hydrolyze protein • Treat with phenylisothiocyanate (PICT) • Separate derivatized AA’s by HPLC +

  13. Protein Sequencing (Edman Degradation) 1) 2) Repeat Trifluoroacetic acid 3) + Can sequence in 30 to 60 AA’s from N-terminus

  14. Generate Proteolytic Fragments • Endopeptidases • Typsin cleaves at COOH end of Lys and Arg • Chymotrypsin cleaves at COOH end of Phe, Tyr, Trp • Chemical Cleavages • Cyanogen Bromide cleaves at COOH end of Met • Generate overlapping fragments • Sequence individual fragements and piece together sequence

  15. 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

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