| Assay Method Information | |
| | Inhibition Against Cytochrome P450 Isoenzymes |
| Description: | Table 9: The inhibition of the test compound against different subtypes of the human cytochrome P450 isoenzyme was determined. The test compound, a standard inhibitor (100×final concentration) and a mixed substrate working solution were prepared; the microsomes frozen in a refrigerator at −80° C. were taken out and thawed. 2 μL of a solution of the test compound and the standard inhibitor was added to corresponding wells, and 2 μL of a corresponding solvent was added to a non-inhibitor control (NIC) well and a blank control (Blank) well; then, 20 μL of a solution of mixed substrate was added to corresponding wells except for the Blank well (adding 20 μL of PB to the Blank well); a human liver microsome solution (labeled with the date after use and immediately putting back to a refrigerator) was prepared and then added to all wells at 158 μL per well; the sample plate was put into a 37° C. water bath for pre-incubation, and then a coenzyme factor (NADPH) solution was prepared; after 10 min, the NADPH solution was added to all the wells at 20 μL per well, and the sample plate was shaken to mix well and then incubated in a 37° C. water bath for 10 min; at corresponding time points, 400 μL of cold acetonitrile solution (internal standard: 200 ng/mL tolbutamide and labetalol) was added to stop the reaction; after being mixed well, the mixture in the sample plate was centrifuged at 4,000 rpm for 20 min, and proteins were precipitated; 200 μL of supernatant was collected and added into 100 μL of water, and the mixture was mixed well and then analyzed by LC/MS/MS. |
| Affinity data for this assay | |
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