| Assay Method Information | |
| | Aromatase Inhibition Activity Assay |
| Description: | This fluorescence-based assay measures the rate at which recombinant human aromatase (baculovirus/insect cell-expressed) converts the substrate 7-methoxy-trifluoromethylcoumarin (MFC) into a fluorescent product 7-ethynyl-trifluoromethylcoumarin (HFC; λex = 409 nm, λem = 530 nm) in a NADPH regenerating system. Briefly, concentrated stock solutions of test compounds were prepared in acetonitrile. Hundred microliters of samples containing serial dilutions of test compounds (dilution factor of 3 between samples) and cofactor mixture (0.4 U/mL glucose-6-phosphate dehydrogenase; 16.2 μm NADP+; 825 μm MgCl2; 825 μm glucose-6- phosphate; 50 μm citrate buffer, pH 7.5) was prepared in a 96-well plate. After incubating the plate for 10 min at 37 °C, 100 μL of an aromatase/P450 reductase/substrate solution (105 μg protein/mL enzyme; 50 μm MFC; 20 mm phosphatebuffer, pH 7.4) was added to each well. The plate was covered and incubated for 30 min at 37 °C. Seventy-five microliters of 0.5 m Tris base was then added to stop the reaction, and the fluorescence of the formed de-methylated MFC was measured with a plate reader (Spectra Max Gemini; Molecular Devices, Sunnyvale, CA, USA). |
| Affinity data for this assay | |
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