Western Blotting problems

Blitzkrieg mediablitz at home.com
Sat Oct 28 13:52:21 EST 2000

1. check the current during transfer
2. check the temperature during transfer
3. make sure you equilibrate PVDF membrane with methanol
4. check pH of transfer buffer
5. remove all air bubbles in sandwich
6. try rinsing the gel in transfer buffer before loading onto transfer

Ralf Kölling <ralf.koelling at uni-duesseldorf.de> wrote in message
news:B610F15D.32A%ralf.koelling at uni-duesseldorf.de...
> 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
> 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
> 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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