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| | Radioligand SPA Binding Assay |
| Description: | To measure the ability of test compounds in the present invention to bind to the human EP3 receptor, and therefore have the potential to antagonize PGE2 activity, radioligand displacement assays were performed. Compound affinity was expressed as a Ki value, defined as the concentration of compound required to decrease [3H] PGE2 binding by 50% for a specific membrane batch at a given concentration of radioligand.Test compounds were half log serially diluted in 100% DMSO (J. T. Baker #922401). 1 μL of each compound was added to appropriate wells of a 384-well plate (Matrix Cat #4322). Unlabeled PGE2 (Tocris Cat #2296) at a final concentration of 1 μM was used to determine non-specific binding. 1 μL of 100% DMSO (J. T. Baker #922401) was used to determine total binding. Millipore EP3 Chem1 membranes (prepared in-house from cell paste derived from the Millipore ChemiSCREEN Human Recombinant EP3 Prostanoid Receptor Calcium-Optimized Stable Cell Line (Millipore Cat # HTS092C, http://www.millipore.com/catalogue/item/hts092c)) were thawed and diluted in binding buffer (50 mM Hepes pH 7.4 (Lonza Cat #17-737), 5 mM MgCl2 (Sigma-M1028), and 0.1% BSA (Sigma A-7409)) to a final concentration of 1 μg/25 μL. 25 μL of diluted membranes were added to prepared compound plates. WGA coated PVT SPA Beads (Perkin Elmer Cat # RPNQ0060) were diluted in binding buffer to a concentration of 4 μg/ul, and 25 μL of the SPA bead mixture was then added to each well for a final assay concentration of 100 μg/well. [3H]-PGE2 (Perkin Elmer Cat #NET428) was diluted in binding buffer to a concentration of 3.375 pM, and 254 was added to all wells for a final assay concentration of 1.125 nM. Plates were incubated for 30 minutes at r.t. (approximately 25° C.) with shaking |
| Affinity data for this assay | |
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