The molecular weights look fine for mmp2 and 9. To fully characterize the digestion bands as MMPs,use organomercurial cleeavage of the proenzyme and 1,10-phenanthroline inhibition of activity. Note that the secreted forms are less likely to be proenzyme forms. Article Matrix metalloproteinase production in regenerating axolotl ...

Article Matrix Metalloproteinase Expression During Blastema Formatio...

Thank you so much for your answer.

How come the pro-MMP retain their gelatinase activity? Aren't they supposedly enzymatically inactive?

Unfortunately, I can't get MMPs from cell extract to work. I harvest the total cell extract as follows:

1x Lysis Buffer (1mL Solution) – Always Prepare FRESH

880uL HNTG (87.8% Final composed of HEPES pH 7.5, Sodium Chloride, Triton X-100, Glycerol)

100uL 10% Triton X-100 (~1.1% Final)

10uL 100x Protease Inhibitor Cocktail Set I (1x Final)

10uL 100mM PMSF (1mM Final)

Any ideas, please?

I’ve gotten good results homogenizing cells in 50 mM Tris base, 0.2% sodium sulfate (SDS), 10 mM N-ethylmaleimide. I homoginize on ice with a probe sonicator. The proenzyme shows gel activity because it is unfolded enough to expose the catalytic site, as long as the coordinately bound zinc is present and calcium is available for substrate binding. This review has a nice diagram and explanation of the proenzyme activity: Article Zymographic techniques for the analysis of matrix metallopro...

I have an additional question, please:

The profile that I got from conditioned media harvested from cells that have been treated with an increasing dose of a broadspectrum MMP inhibitor drug. If you have any idea how to interpret these data?

Did you use 1,10-phenanthroline? The results don’t make sense for an mmp inhibitor.

I used Ilomastat. Is there a cost-efficient MMP inhibitor reagent that you recommend, please?

1,10 phenanthroline is used to treat the samples before running the gel. It is cheap. I don’t use in vivo inhibitors and TIMPs (not cheap) are the only inhibitors I’ve used in vitro. Perhaps another Researchgate user can advise you on cheap inhibitors for cell treatment.

Thank you so much.

Hello Amira,

During Electrophoresis, in a substrate -SDS-PAGE, one will almost always get multiple bands. With 2 step Gel electrophoresis, you will get nice crisp bands of MMPs.

Please refer to the article below

An electroblotting, two-step procedure for the detection of proteinases and the study of proteinase/inhibitor complexes in gelatin-containing polyacrylamide gels- published in Electrophoresis (2001).

BTW, you should also run pure MMP as control (please refer to attached file)

Good luck!

Best regards

Savita

Thank you for your valuable input

I used GM6001 reagent as an inhibitor of MMP's, it works very well, you have to use it to treat your culture before harvesting your medium.

Regards