I recently get a problem when doing semi-dry transfer. After finished SDS-PAGE, I use Towbin buffer with 20% methanol for semi-dry transfer. However, hollow bands (the edge of the band shows little color but the center is hollow at all) appear after developed with BCIP/NBT.
At first I was told that too high protein/antibody level would lead to this problem, but after I stained the gel (after transfer) with Comassie blue, my desired protein was still in the gel. But the weird thing is that I had set the transfer time for 1.5 hours with constant voltage at 15V.
Does anyone also faced the same situation and Is there any suggestion for me?
Protein does not transfer out from the gel
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