Western Blot - How to improve transfer on the sides? (Sep/30/2002 )
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 15 minutes.
-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!!!
Add the height of the paper up and down the PVDF to be sure that the transfer efficiency is enough.