Western blot mystery - possibly a sample preparation problem? - (Mar/28/2012 )

Also by any chance, is your gel assembly tight? or if they are made fresh? If they are dried out, the gels shrink and may become detached from the plate then your samples could be seeping out of the gel instead of running down. Nevertheless, it sounds like you are not getting much protein in the 25 kda to begin with. I think increasing the protein amount to load with fresh SDS probably the next thing to try.

Okay, I found the problem. It was simply that the gels I was making weren't working with my samples. Pre-cast gels work fine, and I get nice, clear, bright bands where Bcl-2 is supposed to be.

Odd thing is, my ladder worked even with the gels I was making. And using fresh SDS didn't help (ALL my reagents for casting gels were purchased within the last 2 months, except for the tris). I'm really not sure why my gels weren't working, since I've made hundreds of gels before in other labs and they've always worked fine. But I also don't really give a damn, I found something that works.

Now I've got a contamination issue in my organ extracts... but hell, that's a way better problem to have than bands not showing up when they should, so I'm still happy.

Thanks for the help everyone!