Topic%2006c%20_%20Electrophoresis%20_%20F10

Topic%2006c%20_%20Electrophoresis%20_%20F10 - Recap:...

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Recap: Native-PAGE histochemical staining of LDH Histochemical Staining for LDH (contains lactate and NAD , plus phenazine methosulfate ( PMS ) and nitroblue tetrazolium sulfate ( NBT ) Lactate + NAD+ PMS (ox) NBTS (ox ) A colorless mixture NADH PMS (reduced) NBTS (reduced) formazan blue precipitate LDH Take home Question. How could you change this mixture to make this activity stain specific for a different dehydrogenase?
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Point 1. Steak LDH is enriched in M form (least charged form, with lowest electrophoretic mobility). [ The relative pIs of M = 8.14 and H = 6.03; hence, the H form is more negatively charged ] Point 2. AMP affinity column preferentially selects the M form of LDH. Point 3. Which H subunit has a larger pI - porcine or heart H? How do you know? M 2 H 2 MH 3 H 4 M 3 H M 4 { Crude LDH fractions Pure LDH Recap: Native-PAGE of LDH
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Agarose gel electrophoresis This is a method used in biochemistry and molecular biology to separate a nucleic acid fragments ( ie DNA and RNA) by length. Nucleic acid molecules are separated through an agarose gel matrix. The charge to mass ratio of nucleic acids are the same (one phosphate group per base), so the velocity of the nuclei acid fragments ( ie separation of the fragments) is based on
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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%2006c%20_%20Electrophoresis%20_%20F10 - Recap:...

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