| Assay Method Information | |
| | Assay for Inhibition of MMP |
| Description: | Serial dilutions of the compound were prepared with 10% DMSO, and 5 μl of each dilution was added to a 50 μl reaction vessel to result in a final DMSO concentration of 1% for each reaction. The enzymes were diluted in 50 mM HEPES buffer pH7.4, 10 mM CaCl2, 0.05% Brij-35, and 1 mM APMA for activation at 37° C. for 2 hours. The enzymatic reactions were conducted in duplicate at room temperature for 30 minutes in a 50 μl mixture containing 50 mM HEPES buffer, pH7.4, 10 mM CaCl2, 0.05% Brij-35, an MMP substrate, an MMP enzyme and a test compound. Fluorescence intensity was measured at an excitation of 328 nm and an emission of 393 nm using a Tecan Infinite M1000 microplate reader. Phosphatase activity assays were performed in duplicate at each concentration. The fluorescent intensity data were analyzed using the computer software, Graphpad Prism. In the absence of the compound, the fluorescent intensity (Ft) in each data set was defined as 100% activity. In the absence of enzyme, the fluorescent intensity (Fb) in each data set was defined as 0% activity. The percent activity in the presence of each compound was calculated according to the following equation: % activity=(F−Fb)/(Ft−Fb), where F=the fluorescent intensity in the presence of the compound. The values of % activity versus a series of compound concentrations were then plotted using non-linear regression analysis of Sigmoidal dose-response curve generated with the equation Y=B+(T−B)/1+10((Log EC50−X)×Hill Slope), where Y=percent activity, B=minimum percent activity, T=maximum percent activity, X=logarithm of compound and Hill Slope=slope factor or Hill coefficient. The IC50 value was determined by the concentration causing a half-maximal percent activity. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |