Western Blotting problems

Farida Safadi-Chamberlain fsafadi at lamar.ColoState.EDU
Sun Oct 29 14:30:28 EST 2000


I had the same problem after i moved to a different lab and started
using new transblotting equipment.  I found out that the foam pads around
the gel and the membrane in the sandwitch are not tight enough to hold the
gel and membrane in place.  I switched to using three pads instead of two
according to the manufacturer's recommendation and my blots started
working.  It could be that your foam pads have gotten old and thinner.=20

Farida Safadi-Chamberlain, Ph.D.
Department of Biochemistry
Colorado State University
Fort Collins, CO 80523
Tel:(970) 491-5149 (lab)     Fax: (970) 491-0494
    (970) 491-0430 (office)

On Sat, 28 Oct 2000, Blitzkrieg wrote:

> 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
> sandwich
>=20
>=20
> Ralf K=F6lling <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 ha=
ve
> > been doing standard Western blotting for ten years without any problems=
=2E
> > 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. "Smearin=
g"
> > usually occurs on certain areas of the membrane, but sometimes the whol=
e
> > 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 methan=
ol,
> > 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 t=
his
> > Western blotting problems could be.
> >
> > Desperately, waiting for an answer
> > Ralf
> >
> >
> > --
> > Ralf K=F6lling
> > Heinrich-Heine-University D=FCsseldorf
> > Germany
> > http://www-public.rz.uni-duesseldorf.de/~koelling/
> >
> >
>=20
>=20
>=20


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