I have been having some trouble doing western blots when using a Midi-sized gel and membrane during the membrane transfer stage. I use the Trans Blot TURBO transfer system from BIO-RAD for transfers. My issue is that I have been consistently getting smeared or "runny" bands near the bottom portion of the membrane where the smaller sized bands are. However, this issue only arises when I am using Midi-sized gels and membranes, and not when using the Mini-sized ones.
-I use; Novex™ 8 to 16 % Tris-Glycine Plus Midi Gels, 20-well (cat#WXP81620BOX) and Trans-Blot Turbo Transfer Pack Midi format 0.2um PVDF (cat#1704157)
-I do the transfers on the Trans-Blot Turbo Transfer System (cat#1704150) and the transfer settings are 2.5A 25V for 3 minutes.
My hunch was that there was too much transfer buffer between the membrane and gel, but I've rolled out as much I can prior to transfer and it hasn't made much difference. I also can't explain why the issue only occurs when using the larger sized membranes. I've included pictures of the band smearing/running. Any help would be greatly appreciated.
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