| Assay Method Information | |
| | Time-Dependent Inhibition of Enzymatic Activity of Site of Metabolism of Midazolam in Human Liver Microsome CYP3A4 |
| Description: | Table 7: A 100 mM PBS buffer was prepared. A 0.25 mg/mL microsome solution, a 7.5 mM MgCl2 and a 5 mM NADPH solution were prepared using the buffer. A 30 mM stock solution was diluted with DMSO to obtain 30 mM, 10 mM, 3 mM, 1 mM, 0.3 mM, 0.1 mM, 0.03 mM and 0 mM serial solutions I, which were further diluted 200-fold with phosphate buffer (PBS) to obtain serial test solutions II (150, 50, 15, 5, 1.5, 0.5, 0.15 and 0 μM). A midazolam working solution was diluted with PBS to 15 μM.The serial test solutions prepared above were well mixed by shaking and aliquoted in 20 μL portions into corresponding reaction plates (+NADPH, T0 and -NADPH groups were set). Three parallel tests were set. 40 μL of the liver microsome working solution was added to each 96-well plate. 20 μL of a corresponding substrate solution was added to the TO plate. 20 μL of NADPH was added to the TO plate and +NADPH group. A timer was started, and the plates were incubated in a water bath at 37° C. After 30 min of incubation, the TO plate was taken out, and the reaction was terminated with 250 μL of an internal standard-containing ACN solution. The +NADPH group was supplemented with 20 μL of the corresponding substrate solution, and the −NADPH group was supplemented with 20 μL of the corresponding substrate solution and 20 μL of NADPH. A timer was started, and the plates were incubated in a water bath at 37° C. After 30 min of incubation, the plates were taken out, and the reactions were terminated with 250 μL of an internal standard-containing ACN solution. |
| Affinity data for this assay | |
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