Attached is my gelatin zymography for 4T1 breast cancer cells. I don't see any banding for MMP2 (around 65-72 kDA) or MMP9 (around 85-92 kDA). In my opinion, the bands above 100 kDa are MMP complexes, and the bands below 50 kDa are MMP1a/b (45-55 kDa) or perhaps MMP14, 15, or 24 (55-65 kDa)?
It just seems very odd that in a gelatin zymography I don't see bands for the gelatinase, but I do for some other MMP's that degrade gelatin as their secondary substrates....Unless those bands are indeed MMP2/9, just at very different sizes than expected. Any tips or input?
indeed strange.. what % acrylamide did you use? an aggregate is possible .
the best is to incorporate a standard MMP in all your runs to avoid these dilemmas
As rightly suggested by Dr Fournier, use MMP standards or alternatively a MMP2/MMP9 positive cell line.
You suggest that bands above 100 kDa are MMP complexes: strange? .Because MMP-MMP complexes should be above > 150 kDa, which it is not the case. Moreover, in the murine system, lipocalin-2 (NGAL in the human system) does not contain the unpaired cysteine that can form the proMMP-9/NGAL complex in humans: in other words, there is no lipocalin/MMP-9 complex around 120 kDa.
Are you sure about the size of your standard markers ? may be the explanation is here.......
It is recommended to make a positive control as a reference. Different gels have different separation effects. Degraded or impure samples can also lead to such results. In addition, the incubation buffer needs to contain the cofactors necessary for the gelatinase reaction to occur.
Tyler Panzner Hello, were you able to solve this problem? I see a similar thing in MDA-MB-231 cells.
Thank you
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