| Assay Method Information | |
| | CYP450 Enzyme Inhibition Assay |
| Description: | Using mixed human liver microsomes as a CYP 450 enzyme source, specific probe substrates of respective CYP isoenzymes (2 substrates for CYP 3A4) are incubated respectively with test compounds of a series of concentrations in the presence of cofactor NADPH. LC-MS/MS is used to determine the amount of metabolites generated by a probe substrate in the incubation system, the IC50 values of the test compounds on specific isoenzymes/substrates are calculated, and their inhibitory effects on the activities of human liver microsomal cytochrome P450 isoenzymes are evaluated. During the experiment, 49 μl of PBS, 50 μl of probe substrate, and 50 μl of human liver microsomal working solution are sequentially added to the incubation system, then added with 1 μl of the test compound working solutions of various concentrations and mixed well. After 5 min of pre-incubation at 37° C., 50 μl of NADPH is added to start the reaction. After incubation for the corresponding time, an appropriate amount of glacial acetonitrile containing an internal standard is added to terminate the reaction, vortexed and mixed well, and centrifuged to take the supernatant, and the supernatant is injected into LC-MS/MS to detect the amount of metabolites generated by the probe substrate. |
| Affinity data for this assay | |
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