| Assay Method Information | |
| | Inhibition of Enzymatic Activity of Site of Metabolism of Testosterone in Human Liver Microsome CYP3A4 |
| Description: | Table 6: I. Experimental Materials and Instruments1. Phosphate buffer (20×PBS, purchased from Sangon);2. NADPH (ACROS, A2646-71-1);3. Human liver microsome (Corning Gentest, Cat No. 452161, Lot No. 905002, Donor36);4. ABI QTrap 4000 LC-MS System (AB Sciex);5. ZORBAX Extend-C18, 3×50 mm, 3.5 m (Agilent, USA);6. CYP probe substrate (testosterone, Vocko, CAS No. [58-22-0]/75 μM) and positive control inhibitor (ketoconazole, SIGMA, Cat No. K1003-100MG).II. Experimental ProceduresA 100 mM PBS buffer was prepared. 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. A30 mM stock solution was diluted with DMSO to obtain 30 mM, 10 mM, 3 mM, 1 mM, 0.3 mM, 0.03 mM, 0.003 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, 0.15, 0.015 and 0 μM). A testosterone working solution was diluted with PBS to 375 μM. 40 μL of a 0.25 mg/mL microsome solution prepared in 7.5 mM MgCl2 and 20 μL of each of the 375 μM testosterone working solution and the compound working solutions (150, 50, 15, 5, 1.5, 0.15, 0.015 and 0 μM) were measured out and well mixed. Ketoconazole at the same concentration was used in place of the compound as a positive control group. A 5 mM NADPH solution was simultaneously pre-incubated for 5 min at 37° C. After 5 min, 20 μL of NADPH was added to each well to start the reaction, and the system was incubated for 30 min. After 30 min, 250 μL of internal standard-containing acetonitrile was added to all the samples. |
| Affinity data for this assay | |
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