| Assay Method Information | |
| | Fluorometric CYP450 Enzyme Inhibition Assay |
| Description: | Test compounds were prepared in 100% DMSO or 100% MeOH and did not exceed a final concentration of <0.2% in the final reaction. A 100 mM sodium phosphate buffer was prepared and adjusted to pH 7.4. In a separate falcon tube, a 2 enzyme/substrate (E/S) solution was prepared in phosphate buffer. The final concentration of CYP2D6 (CORNING ) and 7-amido-4-methyl coumaric acid (AMMC) was 10 nM and 4 uM, and CYP3A4 (CORNING ) and 7-benzyloxy-4-(trifluoromethyl) coumarin (BFC) was 20 nM and 40 uM, respectively. In a separate falcon tube, a 2 NADPH regenerating system (NRS) was prepared in phosphate buffer. The final concentration for each component in the assay was as follows:CYP2D6 assay: 0.008 mM NADPH, 3.3 mM glucose 6-phosphate, 0.4 U of glucose-6-phosphate dehydrogenase per mLCYP3A4 assay: 2.45 mM NADPH, 24.7 mM glucose 6-phosphate, 1.25 U of glucose-6-phosphate dehydrogenase per mLBoth enzymatic assays were conducted in a 96-well microtiter plate (Black, CORNING COSTAR ) with a final volume of 100 uL. Preparation of the plate began with the addition of 74 uL of the E/S in the first well, and 50 uL to all subsequent wells (from 2-11). The test compounds (1 uL) were dissolved in the first well to give the first row a final volume of 75 uL. A 1:3 serial dilution of the test compound was conducted by removing 25 uL from the first well and diluting it with the second and so forth until the tenth row. Final concentrations yielded a range from 200 uM-0.01 uM. Well no. 11 contained no inhibitor, and well no. 12 contained no enzyme. Both were used as controls for background fluorescence. The plate was incubated for 30 min at 37 C. After incubation, the reaction was initiated by the addition of 50 uL of the 2 NRS to each well. |
| Affinity data for this assay | |
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