Western Blues II
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...
"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
> there should be methanol in your transfer buffer anyway... but sometimes
> 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.
> > transfer, some of the protein (the prestained marker, anyway) does leave
> > gel, but does not bind to the nitrocellulose membrane. I have
> > 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
> > it for about 30 seconds in the transfer buffer before it will stay
> > on its own. Even after the transfer, when I try to stain the membrane
> > Ponceau solution, it will still float on top of the solution. Does it
> > like the membrane might have something to do with the problems I've been
> > having?
> > Thanks!
> > Steve
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