I did a perk and terk blot recently to test an agonist, and got these really intense black bands on the 800 channel for pERK, that then turned bright white when I put the images in black/white. The densitometry values around the white bands were negative (i.e. -7.09E0) and made the values for the bands much lower than they have been on previous blots with these antibodies. I'm running again to see if the issue resolves itself, but I'm stumped as to what these bands are, and how to specifically avoid them.

I used biorad tgx 4-20% gel for running and used their criterion system for transfer at recommended settings. The blot was blocked 1hr in odyssey licor BB and the antibodies diluted 1:1000 each in BB. I incubated O/N at 4C, washed with 0.1% PBST, and used licor ms 800 and rb 680 at 1:20,000 for secondary diluted in BB. The primaries were co-incubated, and the secondaries were incubated separately for 1 hr each, with washing in between. I scanned at intensity of 5, although all intensities had these bands.