Western Blotting problems

The Grouchybeast thegrouchybeast at hotmail.com
Tue Oct 17 11:06:49 EST 2000

Ralf Kölling wrote:

> I would be very grateful, if anyone could tell us what the reason for this
> Western blotting problems could be.

Question:  if you stain the gel with Coomassie blue or equivalent, do
you see nice clean bands?  I had a problem with smeary blots which
turned out to be due to uneven gels; the ceramic plates from the Hoeffer
Mighty Small tank had become curved.  Most annoyingly, when the plate
was put in one way round it caused the problem, the other way round it
was fine because the apparatus flattened out the curve.

Assuming the gel is running fine in the first place, I would go with Ian
York's guess and suggest overheating of the transfer apparatus.  A
collegue had a similar problem with smeary blots and that was solved by
reducing the power used for the transfer.  He had whacked the power up
to try and get the transfer done more quickly.

If this a sudden problem which has appeared even though the protocol has
remained consistant, I would suspect a fault in either the transfer
apparatus or the power pack.  If it's a programmable power pack, check
no-one has been messing with the programs.

> Desperately, waiting for an answer
> Ralf


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