| Assay Method Information | |
| | Mu-opioid (hMOP) Receptor Binding Assay |
| Description: | The human μ-opioid (hMOP) receptor binding assay was performed as an filtration-based radio agonist binding assay. Cell membranes of transfected CHO cells (10 μg) were incubated for 60 min at 25° C. with 0.7 nM [3H]DAMGO in the absence or presence of the test compound in a buffer containing Tris 50 mM, MgCl2 5 mM, Saponine 10 μg/ml in a final volume of 100 μl in a 96 well plate. Nonspecific binding was determined in the presence of 10 μM DAMGO.Following incubation, the samples were filtered rapidly over filter plates (GF/C). Filters were washed six times with 0.5 ml of ice-cold washing buffer and 50 μl of Microscint 20 (Packard) was added in each well. The plates were incubated 15 min on an orbital shaker and then counted with a TopCount™ for 30 sec/well.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]DAMGO-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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