Topic%2006b%20_%20Electrophoresis%20_%20F10

Topic%2006b%20_%20Electrophoresis%20_%20F10 - Native - PAGE...

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Native - PAGE mobility charge/mass small = fast low negative charge = slow + + + + large = slow high negative charge = fast + +
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Histochemical stains: formation of color that depends on enzymatic activity highly specific: detection of one enzyme in a mixture example: LDH Visualization of Proteins in Native-PAGE lactate + NAD + pyruvate + NADH + H + e e phenazinemethosulfate nitroblue tetrazolium (ppt)
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Bovine LDH : analysis by Native-PAGE LDH: M (muscle) and/or H (heart) subunits (isomers) similar MW M ~ H ~ 35,000 Daltons different p I M ~ 8.17 H ~ 6.02 MW tetramer 140,000 Daltons Five possible combinations (isozymes) M 4 M 3 H M 2 H 2 MH 3 H 4 p I = 8.17 7.63 7.10 6.56 6.02
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Native-PAGE Analysis of Bovine LDH Net charge at gel pH 8.8 M 4 8.17 barely negative (lowest mobility) M 3 H 7.63 M 2 H 2 7.19 MH 3 6.56 H 4 6.02 most negative (highest mobility) MWs are the same; mobility differences are due to charge differences
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M 1 MATLKDQLIQNLLKEEH-VPQNKITIVGVGAVGMACAISILMKDLADEVALVDVMEDKLK
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This note was uploaded on 11/21/2010 for the course MCB 120L 69059 taught by Professor Fairclough during the Fall '10 term at UC Davis.

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Topic%2006b%20_%20Electrophoresis%20_%20F10 - Native - PAGE...

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