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| | Radioligand Binding Assay |
| Description: | Briefly, ~5μg of membranes were incubated at 30 °C for 60 min with [3H]CP55940 (147.9 Ci/mmol; PerkinElmer Life Sciences) or [3H]SR141716A (43 Ci/mmol; PerkinElmer Life Sciences) in a total volume of 200 or 500 μl of TME buffer (25 mM Tris-HCl, 5 mM MgCl2, and 1 mM EDTA, pH 7.4) containing 0.1% fatty acid-free bovine serum albumin. At least nine radiolabeled-ligand concentrations were used to determine Kd values of the receptors. Nonspecific binding was determined in the presence of 1 μM unlabeled ligand. Reactions were terminated by filtration with a Brandel cell harvester through Whatman GF/C filter paper (Brandel Inc., Gaithersburg, MD) followed by four washes with ice-cold TME buffer to remove unbound radioactivity. Radioactivity was measured by liquid scintillation counting. |
| Affinity data for this assay | |
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