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 tissue homogenate 1000 g Pellet unbroken cells nuclei chloroplast transfer supernatant transfer supernatant transfer supernatant 10,000 g100,000 g Pellet mitochondria Pellet microsomal Fraction (ER, golgi, lysosomes, peroxisomes) Super. Cytosol, Soluble enzymes

5. Chromatography

6. Gel Permeation Chromatography

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

8. Affinity Chromatography Add excess ligand

9. 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

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

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

12. Amino Acid Analysis 1)Acid hydrolyze protein 2)Treat with phenylisothiocyanate (PICT) 3)Separate derivatized AA’s by HPLC +

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

14. Generate Proteolytic Fragments Endopeptidases Typsincleaves at COOH end of Lys and Arg Chymotrypsincleaves at COOH end of Phe, Tyr, Trp Chemical Cleavages Cyanogen Bromidecleaves 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. M atrix A ssisted L aser D esorption I onization T ime o f F light (MALDI-TOF)