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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