Western Blotting problems

Dr. Hiranya S. Roychowdhury hroychow at nmsu.edu
Mon Oct 16 20:40:07 EST 2000


I believe that NC goes bad if exposed to light for too long. As for what
constitutes too long is anybody's guess. Barring this little non-obvious
thing, did you talk to the suppler of the NC?
Also, did you check the conducting grid in the transfer apparatus? If the
current across the sandwich is not even throughout the surface area, you
will get waht you are describing. It is not uncommon to find the wire
making up the two grids broken. Even Pt wires can thin and break following
prolonged usage.



At 05:54 PM 10/16/00 +0200, Ralf Kög==lling 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/
>
>
>
>
Dr. Hiranya Sankar Roychowdhury
College Asst. Prof.
Molecular Biology,
Dept. of Chemistry & Biochemistry	
Box 30001 - 3MLS
New Mexico State University
Las Cruces, NM 88003

Lab: (505) 646 4722
Office: (505) 646 8256
hroychow at nmsu.edu


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