MMP9 protein zymography - (Jan/04/2007 )

Hi everyone

I want to study the effect of a drug on MMP9. I'm trying to do protein zymography. I'm following this protocol but i'm having a problem:

!- I treat the cells in serum free medium for 24 hours.
2- I collecet 1 ml conditioned media and spin it to get rid of the cell depris.
3- I concentrate the media from 1 ml to about 1 ul using amicon ultra-5 centrifugal tubes.
4 I froze my samples at -80 overnight.
5- then next day i ran a 10% gelatin zymogram and followed the manufacturer protocol for washing and staining.
6- i obtained only 1 band and 11 samples failed to give any bands

i appreciate any help and suggestions.

thanks

did you check for enzyme activity immediately before running the zymography gel?

no i did not,, is there a protocol to do that ?

thanks

after you thaw your protein solution you should assay for the enzyme for which you are looking. after confirming the presence of the enzyme then you run the zymography gel. hopefully, the enzyme will renature in the gel and then you will see clear bands when you process. but if there is no detectable activity then you probably won't see anything on the zymogram.

as for how to assay? the assay conditions and substrate depends on the enzyme.

A guy here in the lab uses around 5 ml of conditioned media and concentrates it by dialysis membranes, he got beautiful bands is the zymograms! Maybe the concentrations of enzymes also matters.