Hi! I would like to analyze the activity of MMP2 and MMP9 in extracts from hippocampal tissue. I tried to do it several times but I can't get any bands. I followed Abcam's protocol, which I attached just in case. I wonder if maybe there's not enough protease activity because there's not enough proteins. I tried loading 5ug, 10ug, 15ug, 20ug, 40ug and 50ug of proteins. Does anybody have any advise?
Dear Colleague:
It is very difficult to perform a gelatin zymography for brain protein extract. I tried that method for two years and never worked. Finally, the solution for that problem was to incubate 500 ug of protein with 500 uL of gelatin Sepharose beads (GE Healthcare, catalog no. 17-0956), and then I recovered the MMPs by centrifugation. Then you must to charge all the eluted fraction to the zymography gels. If you have enough tissue, I strongly recomend you to use 1000 ug of protein extract. Remember that you can´t use DTT or B-mercaptoethanol (or any reducting agents) and EDTA or EGTA. You can find a more detailed protocol in my article:
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Best regards
Have you tried chemically activating the proenzyme, or pre-proenzymes found intracellularly? I use 1mM 4-aminophenyl-mercuric acetate (APMA) overnight at 37C in my tissue extracts. This works well with spinal cord and may work with brain. I used homemade substrate gels, but the buffers were the same as in your Abcam system. They don’t suggest the APMA because their method is for secreted enzyme. The APMA ref is cited in the attached paper .
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