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?
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