| Assay Method Information | |
| | [3H]-Radioligand Binding Assay |
| Description: | After thawing, membrane homogenates were resuspended in the binding buffer (8.5 mM HEPES, pH 7.4, 1.3 mM CaCl2, 1.2 mM MgSO4, 118 mM NaCl, 4.7 mM KCl, 4 mM NaHCO3, 1.2 mM KH2PO4, 11 mM glucose) to a final assay concentration of 6.4 μg (OX1) or 1.4 μg (OX2) protein per well. Saturation isotherms were determined by the addition of various concentrations (0-30 nM) of [3H]-4-(2,6-difluoro-4-methoxybenzyl)-2-(5,6-dimethoxypyridin-3-yl)-2H-1,2,4-benzothiadiazin-3(4H)-one 1,1-dioxide (Christopher et al, MedChemComm., 2015, 6, 947-955) in a total reaction volume of 250 μL for 90 min at rt. At the end of the incubation, membranes were filtered onto a 96-well GF/B filter pre-incubated with 0.5% polyethylenimine, with a Tomtec cell harvester and washed 5 times with 0.5 mL distilled water. Non-specific binding (NSB) was measured in the presence of 3.33 μM [4-(5-chloro-1,3-benzoxazol-2-yl)-7-methyl-1,4-diazepan-1-yl][5-methyl-2-(2H-1,2,3-triazol-2-yl)phenyl]methanone (Wertz et al, Angew. Chem. Int. Ed., 2011, 50, 11511-11515). Radioactivity on the filter was counted (1 min) on a Microbeta radiometric plate counter (Perkin Elmer) after addition of 50 μL of scintillation fluid (LabLogic: Part #SG-BXX-14). For competition binding experiments, membranes were incubated with [3H]-4-(2,6-difluoro-4-methoxybenzyl)-2-(5,6-dimethoxypyridin-3-yl)-2H-1,2,4-benzothiadiazin-3(4H)-one 1,1-dioxide at a concentration equal to the KD value of the radioligand (1.5 nM for OX1 and 0.75 nM for OX2 receptors respectively) and 10 concentrations of the inhibitory compound (between the ranges of 10 μM-0.94 pmol). IC50 values were derived from the inhibition curve and the equilibrium dissociation constant (Ki) values were calculated using the Cheng-Prusoff equation. |
| Affinity data for this assay | |
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