1 / 17

Chapter 3 (part 2)

This chapter delves into the essential processes of protein purification and analysis. Pure proteins are crucial for studying enzyme functions, structural analysis, and determining amino acid sequences. Key steps in protein purification include assay development, protein source selection, tissue extraction, cell disruption, and subcellular fractionation. Techniques such as differential centrifugation, chromatography (gel permeation, ion-exchange, affinity), and electrophoresis (SDS-PAGE, isoelectric focusing) are outlined for assessing protein purity and characterization. The chapter also covers methods for amino acid analysis and protein sequencing.

lacey-allison
Télécharger la présentation

Chapter 3 (part 2)

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Chapter 3 (part 2) 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)10-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. large Decreasing MW small 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 fragments 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

  16. Proteomic Analysis

  17. Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF)

More Related