| Assay Method Information | |
| | Receptor Binding Assay |
| Description: | Cell membrane homogenate from Human embryonic kidney HEK-293(T) cells expressing recombinant human prostanoid EP4 receptor was used to perform Prostanoid receptor radioligand binding assays.The competition reaction was initiated by incubation of membrane protein homogenate (20 Mg protein) for 120 min at 22° C. with 0.5 nM [3H]PGE2 ligand in the absence or presence of the test compound in a buffer containing 10 mM MES/KOH (pH 6.0), 10 mM MgCl2 and 1 mM EDTA.Non-specific binding was determined in the presence of 10 μM PGE2 (the corresponding non-radioactive prostanoid). Affinity of the compound binding to hEP4 receptor was measured by displacement of the radiolabeled ligand in the presence of varying doses of tested compound.Incubations were terminated by rapid filtration under vacuum through glass fiber filters (GF/B, Packard) presoaked with 0.3% PEI and washed several times with ice-cold 50 mM Tris-HCl using a 96-sample cell harvester (Unifilter, Packard).The filters were dried and the residual radioactivity bound to the individual filters determined with addition of scintillation cocktail (Microscint 0, Packard) by a scintillation counter (Topcount, Packard).The results were expressed as a percent inhibition of the control radioligand specific binding. |
| Affinity data for this assay | |
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