I am trying to detect MMP-2 and MMP-9 activity in Human aortic vascular smooth muscle cells. I used conditioned media (both directly and concentrated media). I am getting band for both Pro and active MMP-2, whereas I could not find any band for MMP-9. I run the 10% SDS-PAGE gel with 1% gelatin and then incubate at 37C temp for 24hrs. I am using coomassie blue for staining the gel. Can anyone tell me how to solve this issue?
The description factor you gave is too small to find out why. If there are many reasons for the detection, such as MMP-9 detection antibody specificity is not good. It is recommended to set a positive control. If it is an antibody problem, please replace the antibody. Attached to the recommended MMP-9 antibody.
https://www.creative-diagnostics.com/MMP9-antibody-251316-144.htm
When I worked with MMP-9 we obtained better results using an enrichment step, incubatin the tissue (brain tissue in my case) with gelatin-sepharose beads. Also, we used 0.1% gelatin for the SDS-PAGE, we had an overnight renaturation incubation and a longer (72h) digestion period at 37°C. Maybe you can try some of these changes. If you need more detailed information let me know
You may try to use MMP-2 positive cell extracts/sample and look for some papers regarding it. You may try with different antibody.
Hi Javier Pizarro-Bauerle many thanks. the tips you mentioned could be useful for my experiment, I will try these next time.
Jiang Kunpeng and Mohammad Mamunur Rashid thank you very much. But I dont use antibodies for MMP activity assays. thank you again for your response.
Rizwana
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