I am working with a serine protease. After purification, I ran a degradation assay with casein, and it appeared to work great twice in a row (first picture representative of both attempts, first lane is enzyme standard, second lane is casein standard, after that are chronological time points). After that, the assay appeared to show the protease band looking like it's being degraded (second picture). I started running the reaction without casein, and seeing the same thing (third picture). Has anyone seen anything like this? I have tried over and over again, and keep getting this same result. I have tried using new buffer, repurfying in case it went bad real quick, purified a different way to see if there are maybe contaminants I can't see (but that doesn't explain the initial assays). I think it's also very strange that the second time point has a thicker band than the first. I am quenching 10 uL reaction into 5 uL 5x loading dye (which has been made fresh), running on a 12% gel (which had SDS), running buffer has SDS (all has been made fresh). I can't figure it out and I'm out of ideas.