| Assay Method Information | |
| | Binding Affinity Assay |
| Description: | Binding affinities for fentanyl derivatives 1 and 6a-e at KOR, MOR, and DOR were determined by competitive displacement of [3H] diprenorphine as previously reported. In a 96-well plate, cell membranes (10-20 μg of protein) and [3H] diprenorphine (0.2 nM) were shaken in Tris-HCl buffer (50 mM, pH 7.4) with various concentrations of test compound at room temperature for 1 h, allowing the mixture to reach equilibrium. Nonspecific binding was determined using the opioid antagonist naloxone (10 μM), and total binding was determined using vehicle in the absence of competitive ligand. After incubation, membranes were filtered through Whatman GF/C 1.2 μm glass fiber filters and washed with 50 mM Tris-HCl buffer. The radioactivity remaining on the filters was then quantified by liquid scintillation counting in a PerkinElmer Microbeta 2450 after saturation with EcoLume liquid scintillation cocktail. Binding affinity (Ki) values were calculated via “One-site-Fit Ki” nonlinear regression analysis using GraphPad Prism software from at least three independent binding assays performed in duplicate. |
| Affinity data for this assay | |
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