| Assay Method Information | |
| | CYP46A1 Enzyme Assay |
| Description: | Briefly, in a 384-well plate format, 10 uL per well of an enzyme-substrate mixture of CYP46A1 (5 uM final concentration) and Testosterone (10 mM final concentration) were dispensed to wells containing test compound. For CYP46A1 titration, 5 uL per well of serially diluted CYP46A1 (concentrations including 10 uM, 5 uM, and 2.5 uM) and Testosterone (concentration of 10 mM) were dispensed. For Testosterone titration, 5 uL per well of serially diluted Testosterone (concentrations including 10 uM, 5 uM, and 2.5 uM) and CYP46A1 (concentration of 5 uM) were dispensed. The plates were centrifuged at 1000 rpm for 30 seconds, and then sealed and incubated at 37 C. for 30 minutes. The plate was removed from the incubator, and then 10 uL per well of NADPH-generating system were dispensed to initiate the reaction. The plates were incubated at 37 C. for 15 minutes, added with 80 uL per well of 100% methanol consisting 1 ng/ml diclofenac as internal standard, and then transferred for HPLC-MS. |
| Affinity data for this assay | |
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