| Assay Method Information | |
| | AR Binding Assay |
| Description: | AR in LNCaP cytosol was used for determining the binding affinity of test articles and the reference compound progesterone (Sigma, Cat: E2785, St. Louis, MO). IC50s were determined using 8 concentrations/compound. Cytosol was plated at 200 μg/well (100 μL) into a 96-well conical polypropylene plate (Agilent, Cat: 5042-1385, Santa Clara, CA) and mixed with 3 μL of test compound. After adding 100 μL of 3H-methyltrienolone (PerkinElmer, Cat: NET590250UC, San Jose, CA), the plate was sealed and shaken at 300 rpm at 4 C. for 24 hours. Post incubation, 100 μL of radioligand adsorption buffer containing 10 mM Tris-HCl, pH 7.4; 1.5 mM EDTA; 1 mM DTT; 0.25% charcoal; 0.0025% dextran was added to individual well. Plate was shaken for 15 min at 4 C. followed by centrifugation at 3000 rpm for 30 min at 4 C. 150 μL of supernatant was transferred into scint-tube (PerkinElmer, Cat: 6000192) and mixed with 2 mL of Ultima Gold Cocktail (PerkinElmer, Cat: 6013329). Radioactivity was counted using a TriCarb 2910 TR scintillation counter (PerkinElmer). |
| Affinity data for this assay | |
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