protein aggregation (?) in gelatin zymography - (Jun/04/2012 )

Hi,

Whenever I run my tissue extracts on the gelatain SDS gel for zymography, I always end up with most of my sample accumulating at the very top of the 8% separating gel (some weak, fuzzy bands typically appears at approximately the expected size range: 60-90 kDa).

I should mention that others in my lab use the same protocol as myself without this problem, and I didn't have this problem when I performed the same experiment at another institution! My standards (recombinant protein and conditioned media from a cell line) run fine. I suspect problems with sample extraction, but again, I used the same sample extraction protocol previously and I didn't have this problem.

Does anyone have any thoughts or suggestions?

Thanks,

Brad

how do you prepare your samples? overboiling can cause aggregation.