Very high molecular weight activity bands in my gelatin zymography?
I'm performing gelatin zymography on my various heart homogenates and extracts. Unfortunately, I'm getting only very faint, or very diffuse, bands at the expected molecular weight. Instead, I'm getting very heavy bands at the very top of the separating gel (8%), at 250+ kDa. I suspect that it is a sample preparation issue, since (a) my positive controls (purified protein and conditioned media) run fine, and (b) loading my samples on other people's gel gives the same bad result (so it is not a problem with the zymography protocol itself).
One additional, frustrating, point: I used the same protocol at another institution, and it worked fine (ie, nice clean bands at the expected size, no high molecular weight bands). Now I am unable to reproduce my earlier results.
Does anyone have any thoughts or suggestions? Has anyone experienced these very high-molecular weight bands? No one in my lab has ever encountered them.