| Assay Method Information | |
| | Cytochrome P450 Isoenzyme Inhibition Activity Test |
| Description: | The test compound, standard inhibitor (100× final concentration) and mixed substrate working solution were prepared; and the microsomes (purchased from Corning Inc) frozen in the −80° C. refrigerator were taken out and thawed. 20 μL of the test compound and standard inhibitor solution were added to the corresponding well position, and at the same time, 20 μL of the corresponding solvent was added to the control well position (NIC) without inhibitor and the blank control well position (Blank) without inhibitor; secondly, 20 μL of mixed substrate solution was added to the corresponding well position except the Blank well position (20 μL of PB was added to the Blank well position); human liver microsome solution was prepared (the solution was put back in the refrigerator immediately after marking the date after use), and then 158 μL of human liver microsome solution was added to all well positions; the sample plate was put in a 37° C.-water bath for pre-incubation, and then a coenzyme factor (NADPH) solution was prepared; after 10 minutes, 20 μL of NADPH solution was added to all wells, and then the sample plate was shaken well, and incubated in a 37° C.-water bath for 10 minutes; at the corresponding time point, 400 μL of cold acetonitrile solution (internal standard was 200 ng/mL tolbutamide and labetalol) was added to stop the reaction; after the sample plates were mixed well, the mixture was centrifuged at 4000 rpm for 20 minutes to precipitate protein; 200 μL of supernatant was added into 100 μL of water, and the mixture was shaken well and sent to LC/MS/MS for detection. |
| Affinity data for this assay | |
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