Western Blues II

Steve Farris biodork30 at hotmail.com
Thu Jul 4 07:07:40 EST 2002


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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