Western blot problem -disappearing proteins!

Jens tinajens at mail.dk
Mon Dec 13 14:15:12 EST 2004


"jscorah" <jscorah at scripps.edu> wrote in message news:41BA3E9D at neo...
> Hi,
> We are having some major (and very strange) problems with our Western 
> blots
> recently
> and I was hoping someone has some suggestions to solve the problem.
>
> When we run Western blot gels the proteins and marker go through the 
> stacking
> gel into
> the resolving gel and start to resolve nicely. After an hour or so of 
> running
> the gel the
> high molecular weight proteins and markers start to disappear from the top
> downwards.
> By the time the dye front has reached the bottom of a long gel (16cm gel) 
> the
> 75kDa
> marker has started to disappear or become very blurred.
>
> If we transfer these gels there are no proteins on the top of the membrane 
> and
> it almost
> looks like a transfer problem (although we know the problem occurred 
> before
> then).
> Since this problem began we have changed everything common to all people
> (including
> acrylamide, SDS and Laemmli running buffer). We have measured the pH of 
> all
> our
> solutions and they all seem fine and we have recleaned all plates. We have
> experienced
> this problem with all percentage gels and it seems to be getting worse 
> over
> time (as if a
> component of a buffer or something is slowly going off).
> We all use different power packs for running gels and either run them at 
> 30mA
> for 3hours
> or 50V overnight (both of these methods still lead to the same problem).
>
> Any advice or previous experience with this kind of problem would be most
> welcome!
> Thanks.
> Jenny
>
> ---


We had a similar problem with SDS-PAGE some years ago. The low-molecular 
weight proteins were not present or very blurred in the last third part of 
the gel. We found out that the SDS were simply not pure enough. Your problem 
could be related, so you might check the purity of the SDS. The specified 
amount of C12 alkyl should be no less than 98%.
Good luck
Jens











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