| Assay Method Information | |
| | Fluorescence Polarization Assay |
| Description: | To determine IC50 values in the fluorescence polarization assay, solutions of test compounds were prepared and preincubated with COMT enzyme as stated above. Enzyme reactions were initiated upon the addition of 5 μL of an 8× mix prepared in assay buffer containing 8 μM SAM (USB catalog # US10601), 16 μM dopamine (Sigma catalog # H8502) and 40 mM MgCl2. After 25 minutes incubation at room temperature, reactions were quenched with 5 μL 250 mM EDTA, pH 82. To quenched reactions, 20 μL of a preformed complex containing S-adenosyl-L-cysteine (SAC) TAMRA tracer (2 mM from Ana spec diluted 1:80,000) and a 1:20 dilution of anti-S-adenosyl-L-homocysteine antibody (mouse monoclonal from Abbott Homocysteine detection kit, catalog #7D29-20) was prepared in assay buffer 11 (Na2HPO4 pH 7.2). Prior to combining with quenched enzyme assays, the SAH antibody/SAC TAMRA tracer complex was preformed at room temperature for 30 minutes while protected from light. Therefore, the fin |
| Affinity data for this assay | |
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