L6-8 - 1 Biochemistry Sixth Edition Chapter 3: Exploring...

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Unformatted text preview: 1 Biochemistry Sixth Edition Chapter 3: Exploring Proteins and Proteomes Berg Tymoczko Stryer Most biological fuids are a complex mixture oF many proteins ( e.g. milk) MALDI-TO can be used to determine MW oF proteins Chpt 3: Well Focus mostly on 3.1 (introduction to protein purication), 3.5 (mass spec), 3.6 (NMR and X-Ray crystallography), with a brieF discussion oF the other sections. Genome =DNA sequence oF an organism static in size Proteome =Proteins expressed by the organism size varies w/ development, cell type, ... much larger than number oF genes 2 Section 3.1: Protein purifcation: Need an assay to track purity Assay example: NADH production Goal: Maximize specifc activity=rate/[protein] total Done by removing other proteins From sample absorbs at 340 nm no absorption at 340 nm DiFFerential centriFugation allows diFFerent cell Fractions to be assayed Salting out allows simple purifcation and concentration oF proteins DiFFerent ammonium sulFate (0.1 to 5 M) cuts causes diFFerent proteins to precipitate Then need to desalt sample: Dialysis water goes into bag MWCO=1-100kD commercially available stir bar 3 Gel fltration chromatography Biggest proteins elute frst your protein elutes! Ion-exchange chromatography elute with NaCl gradient your protein oF interest: net positive beads: overall negative your protein elutes! Cation exchanger Anion exchanger or Sephadex 4 AfFnity Chromatography beads; have G your protein of interest; binds G your protein elutes! HPLC (or PLC) leads to excellent resolution of complex protein mixtures hplc h igh p ressure (p erformance) l iquid c hromatography fplc f ast p rotein l iquid c hromatography resin is more Fnely divided=better separations! Polyacrylamide gel electrophoresis (PAGE) smaller proteins larger proteins Pipette Buffer t o p o w e r su p p ly 5 PAGE crosslinking ( 29 : 1 is typical ) a crosslink makes pores in gel more crosslink=smaller pores SDS has two jobs for PAGE: 1.) Charges up the protein in a (somewhat) non-speciFc way 2.) Denatures the protein Example of a PAGE gel of proteins--Coomassie-blue stained Top of gel Bottom of gel M M=marker lane low MW protein high MW protein 6 PAGE mobility has a semi-logarithmic dependence on MW unknown protein mobility of 0.46 MW of 29kD Isoelectric focusing acidic protein (=low pI) basic protein (=high pI) gel has a pH gradient due to presence of ampholytes pI is the pH at which charge on protein =0 When pH=pI, protein does not electrophorese positive electrode (Early in run) (End of run) 2-D electrophoresis 1st dimension of electrophoresis (=IEF) 2nd dimension of electrophoresis Band has 5 proteins Band has 3 proteins 1 2 3 1 2 3 4 5 7 A real 2-dimensional gel a protein of interest For E. coli >1000 proteins resolved Purication of a protein: step-by-step large decrease Small decrease...
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This note was uploaded on 07/23/2008 for the course CHEM 476 taught by Professor Bevilacqua,philip during the Fall '07 term at Pennsylvania State University, University Park.

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L6-8 - 1 Biochemistry Sixth Edition Chapter 3: Exploring...

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