| Assay Method Information | |
| | Binding Affinity Assay |
| Description: | Human-cloned dopamine D2L receptors stably expressed in C6 rat glioma cells (kindly donated by Professor Roberto Maggio, Università di L'Aquila, Italy) were radiolabeled with [3H]spiroperidol according to Scarselli et al. with minor modifications [Scarselli et al., J. Biol. Chem., 276:30308-14]. The incubation buffer (120mM NaCl, 5.0mM KCl, 5.0mM MgCl2, 1mM EDTA, 50mM Tris-HCl, pH 7.4) contained 100 μg of dopamine D2L receptor membranes, 0.30-0.50nM [3H]spiroperidol (Kd = 0.093 nM) and six to nine concentrations of drug solution in a final volume of 500 μL. The samples were incubated for 120 min at 25 °C, then the incubation was stopped by rapid filtration through Whatman GF/C glass fiber filters (presoaked in 0.5% polyethylenimine for 60 min). The filters were washed three times in 1mL of ice-cold 50mM Tris, 0.9% NaCl, pH 7.4. Nonspecific binding was determined in the presence of 10 μM haloperidol. The radioactivity bound to the filters was measured by liquid scintillation using a LS6500 Multi-Purpose scintillation Counter, Beckman. |
| Affinity data for this assay | |
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