protein passes through membrane!?

Andreas Vogel vogela at uni-muenster.de
Mon Sep 20 02:19:35 EST 1999


Hi all,

I have severe problems with western blotting! I use the standard
protocol which is used in our lab for YEARS, and suddenly I have
problems (and I need this blot, for sure, very, very soon!!!).
The problem is: the proteins doesnt bind to the membrane but passes
through it, as I can see for the prestained standard, which I can found
in the bottom layer of whatman paper! I tried to use two layers of
membranes and shorter blotting times, but is it everytime the same!
The protocol is: semidry blotting in 10 mM CAPS pH 11.0, 10 % Methanol,
Nitrocellulose membrane 0,2 µm, 30 min with 90 mA for a minigel.

Who has an idea what could be the reason for this?

Any comments are welcome,

Andreas


************************************************************************
Andreas Vogel                
Department of Biochemistry      
University of Münster       Tel: +49-251-8332128
Wilhelm-Klemm-Str. 2        Fax: +49-251-8333206
D-48149 Münster/Germany     E-mail: vogela at uni-muenster.de
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