| Assay Method Information | |
| | MMP-9 Activity Assay |
| Description: | Recombinant human MMP-9 was expressed in Sf9 insect cells and purified by gelatin-Sepharose affinity chromatography as described previously16,17. It was activated with 0.01 μM of the catalytic domain of MMP-3 (cd-MMP-3, Calbiochem) in assay buffer (100 mM Tris/HCl, pH 7.4, 100 mM NaCl, 10 mM CaCl2 and 0.01% Tween-20) at 92 ng/μl (1 μM) as a stock solution as described8. Hydrolysis of the substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 (substrate I, R&D Systems Europe Ltd) was monitored by measuring the increase in fluorescence (excitation wavelength = 328 nm, emission wavelength = 392 nm) [Whittaker et al., Chem. Rev., 99:2735-2776]. All assays were performed at 37°C in 100 μl assay buffer. The reaction was started by adding 10 μl gelatinase B stock solution (0.92 ng/μl) to a 100 μl reaction system with a substrate concentration of 10 μM, after which the rate of substrate hydrolysis was monitored. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |