Exp 5 (cont2) - 20130602_173813

8 bc molecules will barely migrate bc molecules have

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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 of sample 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) seeable 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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