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| | EP3 Radioligand SPA Binding Assay |
| Description: | Test compounds were half log serially diluted in 100% DMSO (J. T. Baker #922401). 1 uL 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 uM was used to determine non-specific binding. 1 uL 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 ug/25 uL. 25 uL 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 ug/ul, and 25 uL of the SPA bead mixture was then added to each well for a final assay concentration of 100 ug/well. [3H]-PGE2 (Perkin Elmer Cat #NET428) was diluted in binding buffer to a concentration of 3.375 pM, and 25 uL 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. Radioactivity associated with each well was measured after a 10 hour incubation using a Wallac Trilux MicroBeta plate-based scintillation counter and a normalized protocol at 1 minute read/well. |
| Affinity data for this assay | |
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