| Assay Method Information | |
| | Inhibition Assay |
| Description: | MMP-2: Recombinant MMP-2 expressed in Chinese hamster ovary (CHO) cells was obtained from Merck-Millipore (catalog number PF023-5UG). DQ gelatin from pig skin fluorescein conjugated (MMP-2 substrate) was obtained from Life Technologies (catalog number E12055).Preparation of the MMP-2 for the activity assay: MMP-2 was provided as a stock solution (0.1 mg/mL). The enzyme was diluted in 1× reaction buffer to a final concentration of 5 μg/mL.Preparation of the DQ gelatin (substrate) solution for the activity assay: the solid form of the substrate was dissolved in water in order to obtain a stock solution of 1 mg/mL.Procedure: The enzymatic assays were performed in 96-well microtiter plate, which allowed simultaneous monitoring of multiple reactions. For each reaction, 86.3 μL of 1× reaction buffer (pH 7.6), 1.7 μL of MMP-2 (final concentration 85 ng/mL) and 2 μL of the corresponding new compound were added to each well. A stock solution of new compound was prepared in DMSO (100 mM), and dilutions were prepared from this stock solution with DMSO. Finally, to the mixture contained in each well 10 μL of the substrate were added (final concentration 50 μg/mL). The reaction was incubated for 2 hours at room temperature in an orbital shaker (100 rpm).The inhibitory activity of the new compound was measured fluorimetrically. The excitation and emission wavelengths were 483 and 525 nm, respectively. |
| Affinity data for this assay | |
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