Western Blotting problems

Savita Shah spshah at stanford.edu
Mon Oct 30 19:01:42 EST 2000


A while ago in this thread there was a suggestion of fluffing the old
pads.. 
I recall it as boiling in water for at least 15 min(?)  or until they
look fluffed ;}
I had this problem when I used the PVDF membrane.. Solution.. just do
not use PVDF.. other nitrocellulose membranes work fine

Savita


> > >
> > > 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 KЖlling
> > > Heinrich-Heine-University DЭsseldorf
> > > Germany
> > > http://www-public.rz.uni-duesseldorf.de/~koelling/
> > >
> > >
> >
> >
> >
> 
> ---






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