Western Blues II

Peter Ashton pda2 at york.ac.uk
Thu Jul 4 08:18:45 EST 2002


Nitrocellulose should not need wetting with methanol (in fact, it may even
be soluble in methanol).  You only need to wet PVDF, but it does sound like
the membrane is behaving like an unwetted bit of PVDF...

Pete

"Philipp Wechner" <philipp.wechner at uibk.ac.at> wrote in message
news:3D2447B8.B74E5CC0 at uibk.ac.at...
> try to soak it in methanol for some seconds before you put it into the
buffer
> there should be methanol in your transfer buffer anyway... but sometimes
it
> helps to rinse the membrane in pure methanol first.
>
> what membrane do you use? which buffer?
>
> Steve Farris schrieb:
>
> > Hi everyone!  I have been having many problems with Western blots.
During
> > transfer, some of the protein (the prestained marker, anyway) does leave
the
> > gel, but does not bind to the nitrocellulose membrane.  I have
formulated
> > new buffer, loaded more protein in the gel, I have upped the transfer
> > voltage, and increased transfer time.  Nothing seems to work.  Something
> > tells me that the membrane might be playing a part in this, but I am not
> > sure.  When I try to prewet the nitrocellulose, I have to forcibly
submerge
> > it for about 30 seconds in the transfer buffer before it will stay
submerged
> > on its own.  Even after the transfer, when I try to stain the membrane
with
> > Ponceau solution, it will still float on top of the solution.  Does it
sound
> > like the membrane might have something to do with the problems I've been
> > having?
> >
> > Thanks!
> > Steve
> >
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