Some days, you’re just better off not going back to the original experiment in its entirety.
I opted to return to the original buffer system for some tryptophan fluorescence in vitro previously done in the lab. Up until now, I was using a different buffer that I had optimized for doing fluorescence anisotropy back during the fall. Using the cross-optimized buffer, I could see 20-30% reductions in tryptophan fluorescence (after accounting for the 10% reduction in signal from ligand addition to induce the conformational change).
Now? Either I’ve gotten way too slow at returning the reaction to the fluorimeter for analysis (doubtful), my protein is crapping out (also unlikely as this is a pretty fresh stock), or this buffer just doesn’t play as nicely. I’m getting maybe a 5–10% change in signal upon ligand addition. And I’m like 3 hours into this 4.5ish hour set of samples.