Problems with wet transfer blot - (Oct/10/2012 )
This was run on 7% gel, transferred at 100v for 1 hour. I've never done wet transfer before, but I can tell that the really black borders around the lanes are abnormal. Is it because of protein overloading? In addition, the band that's shown is actually at 70 kDa and the protein of interest should be 160 kDa - 200 kDa. I've tried semi-dry transfer without success so I guess I'll run less protein and wet transfer for overnight. Any suggestions would be welcomed, thanks!
so... have you tried doing a protein stain of your blot? would be easier and more direct way of answering your questions.
just ponceau stain it and see.
Has marker been transfered well to the PVDF membrane?