| Assay Method Information | |
| | CYP17 Inhibition Assay |
| Description: | A solution of 6.25 nmol of progesterone (in 5 μl of MeOH) was dissolved in 140 μl of phosphate buffer (0.05 M; pH 7.4; 1 mM MgCl2; 0.1 mM EDTA and 0.1 mM DTT) and preincubated for 5 min at 37 °C. together with 50 μl of NADPH-regenerating system (phosphate buffer with 10 mM NADP®, 100 mM glucose-6-phosphate and 2,5 units of glucose-6-phosphate dehydrogenase) and inhibitor (in 5 μl of DMSO). Control incubations were performed in parallel with 5 μl DMSO without inhibitor. The reaction was started by adding 50 μl of a membrane suspension diluted 1 to 5 in phosphate buffer (0.8 to 1 mg of protein per ml). After thoroughly mixing the components, the mixture was incubated at 37 °C. for 30 min. The reaction was quenched by adding 50 μl of 1 N HCl. The steroids were extracted with 1 ml of EtOAc. After a centrifugation step (5 min at 2,500 g), 900 μl of the organic phase was transferred into an Eppendorf vessel with 250 μl of the incubation buffer and 50 μl of 1 N HCl and again shaken. After the centrifugation, 800 μl of the organic phase was removed, placed into a new vessel and evaporated to dryness. The samples were dissolved in 50 μl of a water-methanol mixture (1:1) and analyzed by HPLC. |
| Affinity data for this assay | |
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