| Assay Method Information | |
| | Competitive Displacement Assay |
| Description: | Receptor Binding (in vitro Assay) The Ki (binding affinity) for mu-, delta-, and kippa-receptors was determined with a previously described method using a competitive displacement assay (Neumeyer, 2003). Membrane protein from CHO (Chinese Hamster Ovarian) cells that stably expressed one type of the cloned human opioid receptor were incubated with 12 different concentrations of the compound in the presence of 0.25 nM [3H]DAMGO, 0.2 nM [3H]naltrindole or 1 nM [3H]U69,593 in a final volume of 1 mL of 50 mM Tris-HCl, pH 7.5 at 25 C. Incubation times of 60 min were used for [3H]DAMGO and [3H]U69,593. Because of a slower association of [3H]naltrindole with the receptor, a 3 h incubation was used with this radioligand. Samples incubated with [3H]naltrindole also contained 10 mM MgCl2 and 0.5 mM phenylmethylsulfonyl fluoride. Nonspecific binding was measured by inclusion of 10 uM naloxone. The binding was terminated by filtering the samples through Schleicher & Schuell No. 32 glass. |
| Affinity data for this assay | |
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