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| | Binding Affinity Assay |
| Description: | Human-cloned 5-HT1A serotonin receptors stably expressed in HEK293-EBNA cells (Perkin-Elmer, Waltham, MA) were radiolabeled with 1.0nM [3H]-8-OH-DPAT [Fargin et al., J. Biol. Chem., 264:14848-42]. Samples containing 32 μg of membrane protein, different concentrations of each compound ranging from 0.1nM to 10 μM were incubated in a final volume of 500 μL of 50mM Tris-HCl pH 7.4, 5mM MgSO4for 120 min at 37 °C. After this incubation time, samples were filtered through Whatman GF/C glass microfiber filters presoaked in polyethylenimine 0.5% for at least 30 min prior to use. The filters were washed twice with 1mL of ice-cold buffer (50mM Tris-HCl, pH 7.4). Nonspecific binding was determined in the presence of 10 μM 5-HT. The radioactivity bound to the filters was measured by liquid scintillation using the LS6500 Multi-Purpose scintillation Counter, Beckman. |
| Affinity data for this assay | |
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