| Assay Method Information | |
| | Cytochrome P450 Isoenzyme Inhibition Assay |
| Description: | Experimental operation: Firstly, the test compound (10 mM) was subjected to gradient dilution to prepare a working solution (100×final concentration), and the concentration of the working solution was: 5, 1.5, 0.5, 0.15, 0.05, 0.015 and 0.005 mM, respectively, and the working solution of each positive inhibitor of P450 isoenzyme (CYP1A2 and CYP3A4) and its specific substrate mixture (5 in 1) were prepared; and then the human liver microsomes frozen in the −80° C. refrigerator were thawed on ice, and all the human liver microsomes were dissolved, diluted with PB, and a certain concentration of working solution (0.253 mg/mL) was prepared; 20 μL of substrate mixture was added to the reaction plate (20 μL of PB was added to the blank well), while 158 μL of human liver microsomal working solution was added to the reaction plate, and the reaction plate was placed on ice for later use; at this time, 2 μL of various concentrations of the test compound (N=1) and specific inhibitor (N=2) were added to the corresponding well, and the corresponding organic solvent was added to the non-inhibitor (test compound or positive inhibitor) group as a control group sample (a ratio of DMSO:MeOH in the test compound control sample was 1:1, a ratio of DMSO:MeOH in the positive control sample was 1:9); after pre-incubation in a water bath at 37° C. for 10 min, 20 μL of coenzyme factor (NADPH) solution was added to the reaction plate, and incubated in a water bath at 37° C. for 10 min; and 400 μL of cold acetonitrile solution (internal standard was 200 ng/mL tolbutamide and labetalol) was added to terminate the reaction; the reaction plate was placed on a shaker and shaked for 10 minutes; and then centrifuged at 4,000 rpm for 20 minutes; 200 μL of the supernatant was taken and added to 100 μL of water for sample dilution; finally the plate was sealed, shaked and shaked well, for LC/MS/MS detection. |
| Affinity data for this assay | |
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