Western Blues II

Philipp Wechner philipp.wechner at uibk.ac.at
Thu Jul 4 08:03:53 EST 2002


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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