Western Blotting problems

clarosa clarosa at biocomp.unl.edu
Mon Oct 16 12:09:54 EST 2000


I have no idea... I always used semidry, because it worked much better than wet
transfer..

Ralf Kölling wrote:

> 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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