| Assay Method Information | |
| | Gal4-luciferase reporter assay |
| Description: | Briefly, the AR LBD is expressed as a fusion with the Gal4 transcription factor, which binds to the Gal4 reporter DNA. Upon activation with agonist hormone (DHT), the Gal-AR LBD binds to the Gal4 reporter gene, which in turn drives the transcription (and subsequently translation) of the reporter luciferase. The effect of antiandrogens (competitive antagonists) on this process is measured by quantifying the amount of luciferase activity after 24 hours in the presence of varying concentrations of antiandrogens. From these values, an IC50 for each antagonist is calculated. Experimentally, Hela cells were maintained in Dulbecco's modified Eagle's medium H-21 4.5 g/L glucose, containing 10% steroid depleted fetal bovine serum, 50 units/mL penicillin. For transfection, (1×105) cells per well were plated and incubated overnight. A mixture of typically 200 ng of Gal4 responsive luciferase reporter plasmid, 10 ng of β-actin-β-galactosidase internal control, 10 ng of GAL-AR LBD or empty vector control, and 10-100 ng of β-catenin or empty vector control were mixed with 0.5 μL of transfection reagent from BioRad and incubated for 20 min and then plated in 24 well plate triplicates. Cells were induced with 1 nM DHT and 0.1 nM-30 uM test compounds after 3 hr and then incubated over night. Cells were collected, and pellets were lysed in 100 μL of 100 mM Tris-HCl (pH 7.5) containing 0.1% Triton X-100. Luciferase and β-galactosidase activities were measured using the Luciferase Assay System (Promega) and Galacto-Light Plus-galactosidase reporter gene assay system (Applied Biosystems), according to the manufacturer's instructions. |
| Affinity data for this assay | |
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