Hi,

Do you ask about gelatin zymography technique or just about zymogram analyse? Where do you have a trouble? What protocol do you use? In generally zymography is widely used in labs and it is relatively simply technique. Even if you change some reagents you should do it succesfully. The main rule is performing the electrophoresis with SDS but without thermoblot and without mercaptoethanol, then renaturation of MMPs by washing the gel in 2.5% Triton X100 and incubation in 1% Triton for 24 hours. I use coomasie blue R250 in 30% methanol +10% acetic acid and destained with the same buffer but without Commasie. That is all.

Send more particualr description of your troubles.

Regards,

JK

Dear Samet, Jacek above is correct that it is relatively simple to do gelatin zymography.  I would like to add a couple of other points - in that it depends on your sample preparation, but how much enzyme activity you have in your samples, which may mean you need to alter how much sample you add, or change the time of incubation of the gel at 37deg.  So the first thing to work on is - what methodological control are you using? - Can you show that you are detecting activity in a control sample with known activity - If not, it is a method problem.

Are you doing gel zymography (and not in situ zymography or in vivo zymography), and are you using gelatin (and not casein, or some other protein) as the substrate?  Do you have positive controls (i.e. are you running some purified MMP-2, or conditioned medium in one lane)?  Do the controls work?

The most common mistake I see students making with this is that they prepare their samples in standard sample buffer, which contains DTT, 2ME or some other reducing agent, and that will destroy the enzyme activity.

If you can provide more methodological detail, that would be helpful.

Thanks for your interest. I obtain the result via trial-error.

Thanks again...