| Assay Method Information | |
| | Delta-opioid (hDOP) Receptor Binding Assay |
| Description: | The human δ2-opioid (hDOP) receptor binding assay was performed as an filtration-based radio agonist binding assay. Cell membrane homogenates of transfected Chem-1 cells (1 μg) were incubated for 60 min at 22° C. with 0.5 nM [3H]DADLE in the absence or presence of the test compound in a buffer containing 50 mM Tris-HCl (pH 7.4) and 5 mM MgCl2 in a final volume of 200 μl in a 96 well plate. Nonspecific binding was determined in the presence of 10 μM naltrexone.Test compound was added at a 100× concentrated solution in solvent and final assay DMSO concentration was 1% maximum which also served as respective vehicle control.Following incubation, the samples were filtered rapidly under vacuum through glass fiber filters (GF/B, Packard, presoaked with 0.3% PEI) and rinsed several times with ice-cold 50 mM Tris-HCl using a 96-sample cell harvester (Unifilter, Packard). The filters were dried and counted for radioactivity in a scintillation counter (Topcount, Packard) using a scintillation cocktail (Microscint 0, Packard).The results are expressed as a percent inhibition of the control radioligand specific binding. Half-maximal inhibitory concentration (IC50) values reflecting 50% displacement of [3H]DADLE-specific receptor binding are calculated by nonlinear regression analysis and Ki values are calculated by using the Cheng-Prusoff equation. |
| Affinity data for this assay | |
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