Western Blot, How to improve transfer on the sides?
marsupilamiwolfgang at web.de
Mon Sep 30 09:56:22 EST 2002
It seems that you have a different current in the middle of the gel than at
the edges. Possibly this is due to a inhomogenous pressure and / or
You could try 4h at 1/4 of the current you currently :) are using. Do not
rely on voltage settings, since its diffficult to control the height of the
setup in a reproducible way and results might vary also because of that.
There also is a possibility that you "short cut" the gel edges with
overlapping filter paper.
Either make the paper the same size as the gel or use 3 or 4 layers each to
minimize the effect. You then have to increase the voltage or the time, of
course, since the resistance of the stack increases. Better set the same
current as usual and limit the voltage to the initial value, to prevent
overheating, than using ice. You even might to put the setup into a cold room.
At 13:34 30.09.2002 -0000, passardip at ngene.hu wrote:
>I have been starting doing Westerns for 6 months now, and I still have a
recurrent problem I would really appreciate if anybody could help me to
solve. I am transfering my proteins onto PVDF membranes with a semi-dry
blotting method, and I always have a decreased efficiency of transfer on
the samples situated on the edges of my gel.
>Briefly, the procedure used is the following:
>-rehydrate PVDF in MeOH during 2 minutes
>-incubate PVDF and polyacrylamide gel into a solution of 32ml TBS 10x,
80ml MeOH and completed up to 400ml with distilled water. Incubation time
>-apply one blot paper (also soaked in the previously mentioned solution),
then PVDF, then gel, then blot paper.
>-Transfer 1hr at 15V constant. I also apply some ice on top of the
transfer device to prevent overheating.
>Has anyone got the same problems? Has anyone found a solution?
>THANKS A LOT!!!
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