Lecture10 - MCB 110L Spring 2011/18/2011 Prof Qing Zhong...

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Unformatted text preview: MCB 110L Spring 2011 Lecture 10 02/18/2011 Prof. Qing Zhong Gel electrophoresis • Basic principles • Electrophoretic methods • Native PAGE • Activity and protein staining Denaturing gels - SDS-PAGE • Most common method to estimate: › Purity › Subunit mass • Advantage over native gels - separate using only one protein parameter → size • How? › Make all proteins enter gel simultaneously › Give all proteins same charge density › Give all proteins same shape (random coil) SDS-PAGE - components • Must make chemistry of experiment interrogate only size by manipulating conditions: › Sample loading buffer › Running buffer › Gel casting buffers › Discontinuous acrylamide concentrations Buffer components I - SDS • Sodium dodecyl sulfate - anionic detergent • Complexes with protein - approx. one SDS molecule per two amino acids › Overcomes natural charge › Gives all proteins similar mass/charge ratio • Breaks non-covalent bonds › Disrupts subunit interactions › Unfolds protein into random coil • Mobility through electric Feld proportional to mass O S O O O Na Buffer components II - reducing agents • β-mercaptoethanol • Dithiothreotol (DTT) • Reduces/prevents cystine (Cys-Cys disulFde) formation • ¡urther removes secondary and tertiary structure HS H 2 C H C OH C H OH H 2 C SH H 2 C CH 2 OH SH Other notables • Heat - sample only › Generally 95˚C (1-4 minutes) assures/ accelerates unfolding • Glycerol (sucrose/fcoll) - loading buFFer › Increases density of solution to allow loading into wells...
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This note was uploaded on 09/12/2011 for the course MCB 120L taught by Professor Fairclough during the Spring '08 term at UC Davis.

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Lecture10 - MCB 110L Spring 2011/18/2011 Prof Qing Zhong...

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