IUBio

HELP: peptide separation on SDS-PAGE

L. Lintott llintott at shaw.ca
Fri Nov 30 01:05:08 EST 2001


"D.K." wrote:
> 
>  "L. Lintott" <llintott at shaw.ca> wrote:
> >With older BioRad mini's this can be a
> >problem as they tend to leak from the upper chamber.
> 
> Not if they are properly assembled.
> 

> >I always put lots
> >of grease on the gasket to prevent this.
> 
> I thought the whole idea of their design was to get rid
> of disgusting grease :-)

When the holder gets old, it looses its 'spring', even with new gaskets
it doesn't snap in tight enough to prevent leaking.  I think that System
III will stand up a lot better, but I have no money to buy any new
stuff.  You would die laughing if you saw the old 'hand me downs' I have
to work with.    I agree, the grease is disgusting, but effective.


> 
> >I also use a polypetide fixing solution on the gel before I stain
> >coomassie (40% methanol and 10% acetic acid).
> 
> Does not work well for many polypeptides below 5k. Aldehyde
> fixation is a lot more pain but works better.
> 
I found it worked well down to about 2 kDa, but it probably depends on
the particular peptide.  

> >Recently I have
> >switched to using Invitrogen's "Simply Blue" and find that it works very
> >well for the smaller proteins, and is somewhat simpler to use.
> 
> It is simply Bradford's reagent. You can make 1 L of it in ten minutes
> for the price of 60 mg Coomassie and 100 ml conc. phosphoric acid.
> 

Really?  Thats all thats in it?  I will try that out.  Its not nearly so
stinky and saves having to dispose of all that methanol.


> DK




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