| Assay Method Information | |
| | Cytochrome P450 Isoenzyme Inhibitory Activity Assay |
| Description: | Test compounds, standard inhibitors (at 100× final concentration), and mixed substrate working solutions were prepared; the microsomes (purchased from Corning Inc.) stored at −80° C. were taken out and thawed. To the corresponding wells were added 20 μL of the test compound and standard inhibitor solutions. Meanwhile, 20 μL of the respective solvent was added to the No Inhibitor Control (NIC) and blank control wells. Next, 20 μL of mixed substrate solution was added to the corresponding wells, except the blank wells where 20 μL of phosphate buffer (PB) was added. A human liver microsome solution was prepared (immediately returned to the fridge after the date of use was marked). Then, 158 μL of this solution was added to all the wells. The sample plate was placed in a 37° C. water bath for pre-incubation. A co-enzyme factor (NADPH) solution was then promptly prepared. After 10 minutes, 20 μL of NADPH solution was added to all wells. The sample plate was shaken to mix and placed back into a 37° C. water bath for an additional 10 minutes of incubation. At the respective time points, the reaction was terminated by adding 400 μL of cold acetonitrile solution (internal standard at 200 ng/ml of tolbutamide and labetalol). The sample plate was mixed thoroughly and was then centrifuged at 4000 rpm for 20 minutes to precipitate proteins. 200 μL of the supernatant was taken and mixed with 100 μL of water, after which it was sent for LC/MS/MS analysis. |
| Affinity data for this assay | |
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