| Assay Method Information | |
| | MMP Inhibitory Assay |
| Description: | The inhibitory effect of compounds on the rate of cleaving fluorogenic MMP substrate (Enzo, BML-P128) by recombinant human MMP-12 catalytic domain (Enzo, BML-SE138) was carried out by methods known in the art. Briefly, to each well of a 96-well black opaque plate, all the reagents were sequentially added by pipetting, and the final reaction contained 4 nM of recombinant human MMP-12 catalytic domain, 4 μM of fluorogenic MMP substrate, and various concentrations (0.057 nM to 1,000 nM) of tested compound dilutions in HEPES buffer (pH 7.5) containing 10 mM of CaCl2, 0.01% Brij 35 (polyoxyethylene (23) lauryl ether), and 0.1 mg/ml of BSA. The enzyme and compounds were pre-incubated on a shaker to mix in wells. After an hour of mixing, fluorogenic substrate was added to each well. Reaction without enzyme was used as a blank control in the plate. The plate was then fed into a plate reader to measure fluorescence intensity at Excitation/Emission wavelengths of 340 nm/440 nm every 10 mins for at least 1 hour at 37° C. |
| Affinity data for this assay | |
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