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Unformatted text preview: pH 6.8 --> bc molecules will barely migrate bc
molecules have pI around pH of stacking gel --> dont use stacking gel in this lab Use razor to cut in pieces put in eppendorf tube
+ buffer--> assay for E
activity bc native
doesn't kill protein crude W/O stacking gel, large sample cant get thinner but actually get wider
cuz of diffusion so running a gel wo stacking gel, should put a small amount
in SDS, proteins most of the time go right direction bc SDS bind to proteins make them always (-) In Native gel, things can go wrong way. If proteins r + charged--> migrate up Histochemical Stain
LDH Lactate + NAD+ --------> pyruvate + NADH + H+
NADH cant convert back to NAD+ if it
can transfer H+ to somewhere else phenazine methosulfate (oxidized) phenazine methosulfate (reduced)
If it can pass 2H+ to somewhere this step can't see Nitroblue tetrazolium salt Blue insoluble precipitate in the gel (yellow)
Therefore, we use phenozine methosulfate by colliding with NADH n take H+--> unseeable--> use nitroblue
tetrazolium salt picking up H+ of phenazine methosulfate--> seeable blue precipiate
--> no need to cut the getl - The more u let it sit--> the more the bands growing - How many b...
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This note was uploaded on 10/12/2013 for the course MCB 120L taught by Professor Fairclough during the Fall '08 term at UC Davis.
- Fall '08