I am trying to extract matrix metalloproteinase-2 (MMP-2) protein from the cell.
At first, I just used the common protocol (by RIPA kit) for whole cell proteins, but it seemed to be a wrong process as I could not see the right band size of MMP2 by western blot (which would be 72 kDa for inactive form and 66 kDa for active form but what I observed was a 90 kDa band).
So I thought that, since MMP2 is one type of proteinase, so proteinase inhibitor and PMSF those are included in RIPA kit components, should not be added in the extraction reagents.
But so far, I could not find any protocol that could be applied for such protein.
Could any one having experience give me some suggestion for my matter.
Thank you so much for all your help.
Hoang Thi Xoan
Dear Hoang
I would like to know your protocol from plating the cells to lysis. I think best way to evaluate inactive and active MMP-2/MMP-9 is to go for gelatin zymogram, it is an economical and quick assay, you can show the actual activity of MMPs (substrate degradation). Gelatin zymogram may be performed from the cell lysates as well as conditioned media ( media in which treatment is given).
Given the above status, I would like to know your detail protocol of experiment as well as lysis. For further query feel free to mail at [email protected].
would love to share my experimental skills as well as methods.
Regards
Anil
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