So it's been a while since I've done wet transfer (in my graduate lab, we used semi-dry). I'm trying to transfer ATM, a 350-kDa protein, to a PVDF membrane from a 6% Tris-Glycine gel. When I took my apparatus to the cold room to begin the transfer (but before I hooked it up), I noticed that precipitate was forming in the transfer buffer. It was at that point that I realized that I had accidentally made my buffer with 10X Tris-Glycine and 20% methanol, not with 1X Tris-Glycine and methanol. I immediately took it back into the lab, where I set up another apparatus with 1X buffer + methanol and transferred the entire cassette from the first box. I then set up everything else as normal and started the overnight transfer.
So, the big question is whether I messed with the integrity of my experiment. Does exposing the gel to higher concentrations of Tris-Glycine cause any adverse effects to the proteins in the gel, such as leeching? I have a feeling that I'm in the clear, because I realized what I did prior to starting the transfer, but I just want to make sure.