Western Blotting problems

Ralf K ö lling ralf.koelling at uni-duesseldorf.de
Mon Oct 16 10:54:38 EST 2000


Hi,

were are getting desperate - maybe someone out there can help us. We have
been doing standard Western blotting for ten years without any problems.
But, recently we have encountered problems for which we don't have any
explantion. After electrophoretic transfer to nitrocellulose by "wet
transfer" (according to Towbin et al.) in standard blotting buffer (25 mM
Tris, 190 mM glycine, 20 % methanol), we stain the proteins on the blot with
Ponceau S. Instead of the normal protein banding pattern, we often see
"clouds" of staining on the NC membrane ("smear blots" as we call it). It
seems as if the protein doesn't properly bind to the membrane. "Smearing"
usually occurs on certain areas of the membrane, but sometimes the whole
blot is affected. Sometimes the blots are also perfectly o.k. It's
unpredictable. We have considered all sorts of things that might be
responsible for this effect (source of nitrocellulose, purity of methanol,
pads, thickness of the "transfer sandwich") but there has been no consistent
correlation between the problems and any of these things.

I would be very grateful, if anyone could tell us what the reason for this
Western blotting problems could be.

Desperately, waiting for an answer
Ralf

 
-- 
Ralf Kölling
Heinrich-Heine-University Düsseldorf
Germany
http://www-public.rz.uni-duesseldorf.de/~koelling/







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