| Assay Method Information | |
| | Radioligand Binding Assay: |
| Description: | The radioligand EP4 binding assay was performed using ChemiScreen recombinant human EP4 receptor membrane preparations from Millipore, according to manufacturer's instructions. Briefly, membranes prepared from Chem-1 cells overexpressing human EP4 cDNA (Millipore) were mixed with 1.8 nmol.L−1 [3H]-PGE2 and 5 μmol.L−1 unlabelled PGE2 in the presence or absence of various concentrations of testing compounds in binding buffer (50 mmol.L−1 HEPES, pH 7.4, 5 mmol.L−1 MgCl2, 1 mmol.L−1 CaCl2, 0.2% BSA) in a nonbinding 96-well plate, and incubated for 1-2 h at room temperature. Prior to filtration, a GF/C 96-well filter plate was coated with 0.33% polyethyleneimine for 30 min, then washed with 50 mmol.L−1 HEPES, pH 7.4, 0.5% BSA. Binding reactions were transferred to the filter plate, and washed 3 times with Wash Buffer (1 mL per well per wash). |
| Affinity data for this assay | |
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