I am trying to quantify the gelatinase activity of BMDC lysates. The BMDCs are incubated for 24h, the medium R10 is then removed and the cells are scraped in PBS, samples are then sonicated. I am using the Enzcheck gelatinase activity kit, which uses gelatin labeled with flourecin that becomes flourescent upon the cleavage of gelatin. I have not yet been able to detect any gelatinase activity. My positive control (provided in the kit) works fine and I have tried adding NaHCO3 to compensate for any possible pH effects. Any ideas what could be the problem?
I don't know if this might be the problem, but gelatinases are proteins that are secreted in the medium, so if you use cell lysates you should get a very low activity (maybe undetectable also by zymography) or nothing. This might be the problem. As far as I know the only cells that store gelatinases (only MMP-9) are the neutrophils....and also if you are searching for activity, you will not get anything since it is inactive...
yes i am also with Alessandro. As far as I have learned, I will also suggest you to check the gelatinase activity in the medium.
Thanks and please add something if you get more idea
I agree that most of the Gelatinase should be in the media. But there is some in cells as well. I was wondering if while sonicating, the temperature got too high, and cooked the enzymes.
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