Weak Zymography Signals - Mitigating a weak signal for better bands (Jan/07/2011 )
I was wondering if anyone had thoughts on how to increase signal for MMPs in zymography. I understand that one can either add more protein, or incubate the enzymes longer before staining, but are there any other factors that could lead to a reduction in signal?
Normally, I have read that researchers use 1-10 ng of MMP-2 standard in gelatin zymograms and get good bands, however I have found in my application that I need to increase this number to about 30ng. This is after an overnight incubation as well, recommended for higher sensitivity.
This has been my go-to protocol.
Any thoughts are much appreciated!
Hola, only one thing and excuse me if you done it, I remember that the sample buffer for zymograms is withouth reducer and the samples are loading without boiling as the protocol says. Good luck