Hi Kurma,

this is the protocol I used some years ago in order to perform MMP9 zymography, the reference is Gargioli et al., 2008 Nat Med.:

Zymography. MMP-9 activity was determined by gelatin zymography as described in 24 with modifications. Briefly, the samples were separated on 10% polyacrylamide gel with 0.05% gelatin type A from porcine skin (SIGMA G2500). After electrophoresis, washing was carried out for two 30-min periods with 50 mM Tris-HCl, pH 7.2, containing 10 mM CaCl2, 0.02% NaN3 and 2.5% Triton X-100. Then the gels were incubated 18 h at 37°C in the above buffer but with 1% Triton X-100. Gels were stained with 0.1% Coomassie Blue R-250 in 30% ethanol and 10% acetic acid and destained in 30% ethanol and 10% acetic acid. MMP-9 activity was detected as clear bands on the blue background. Enzymes were identified by co-migration with recombinant mouse MMP9 (SIGMA M1552 ).

Good luck

Avoid a common mistake: do not use EDTA to inhibit the proteases in your protein extration sample. Good luck

Dear Cesare & Benjamin,

Thank you very much for your suggestions.

Lysis buffer I used contain 5 M Tris·HCl, (pH 7.45), 2.0 M sodium chloride, 50 mM calcium chloride, and 0.2% Brij-35.

I used 10% gel and in the gel I cannot see two forms of MMP's

(I have attached the gel).

Can you please suggest me possible solution.

Thank you very much in advance

Dear Kurma,

I do see two forms of MMPs..

(I attached your gel with indications)

It also looks like these bands are in the correct size (~66kd and ~92kd for MMP2 and MMP9, respectively).

I think you should destain less your gel and then the background will be more blue and the bends will stand out more

Good luck

Dear Rotem kalev-altman,

Thank you very much for your reply.

About the forms of MMP's I mean active and latent form of MMP's. From the gel I can't see active and latent forms of MMP's.

Your bands are pretty and sharp. Well, I think incubation time has an effect. Possibly, keeping gel for longer time in zymograpay buffer can reduce the bands for latent form. For me it happened when I incubated overnight I had both forms. Upon 2 days of incubation my latent bands became weaker.

You need to destain less. Rest seems good.

can you share you protocol? How much gelatin you used? I am fighting with my mmp9 bands. My gel has sharp mmp2 bands but mmp9 is not visible.