Hello,
I would like to perform zymography on rat tissue;Can anyone suggest me the protocol.
Thanks in advance for your help!!
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.
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