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So I'm running a pretty typical SDS-PAGE setup. Discontinuous gel (20% resolving), fresh sample buffer with BME/SDS, 90 degrees for 10 min before loading, etc. I'm probing with an antibody that recognizes a phospho-serine on my protein of interest, and I'm trying to normalize to the total amount of that protein after stripping and reprobing. The phospho-antibody is polyclonal and the total protein antibody is monoclonal.
The problem: my phospho-serine antibody gives bands exactly where I'd expect (~25 kDa), but the total protein band is all the way up at 250 kDa (no bands at 25 kDa). The only way I know how to explain this is I might need more BME to get rid of disulfide bonds, more SDS, or maybe a different pH in either the stacking or resolving gels. I've already tried not heating before I load, and I've made fresh sample buffer. Any ideas? I'm really stuck here!
