| Assay Method Information | |
| | CYP3A4 Inhibitory Activity Assay |
| Description: | Test compounds, DMSO (negative control), and ketoconazole (positive control) were diluted to 4× final concentrations in water. The standard final Compound concentrations were 37, 111, 333, 1000, and 3000 nM. 12.5 μL of the Compound dilutions were transferred to a white 96-well plate. 1450 μL (enough for a whole plate) of 4× assay buffer (400 mM potassium phosphate buffer (10 mL 1M potassium phosphate buffer: 8.02 mL 1M K2HPO4 +1.98 mL 1M KH2PO4 (1.4 g K2HPO4 +0.27 g KH2PO4 in 10 mL H2O), 32 μM Luciferin-IPA (Promega V9002)) 580 μl of 1 M K3PO4 buffer, 870 μL H2O, 14 μL of 3 mM Luciferin-IPA, and 18 μL of human liver microsome (Sigma M0317-1VL) was made. 12.5 μL of 4× assay buffer was added to each well. For the well of blank control, 12.5 μL of 4× assay buffer without liver microsome was added. The plate was incubated at room temperature for 15 minutes. 2.75 mL NADPH buffer was made as follows: 2.42 mL H2O, 275 μL solution A and 55 μL solution B (NADPH regeneration system, Promega V9510). 25 μL of the buffer was added to each well. The plates were incubated at 37° C. for 11 minutes. 50 μL of luciferin detection reagent (Promega V9002) was added and the plates were incubated at room temperature for 5 minutes. The plate was read with a luminometer. |
| Affinity data for this assay | |
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