| Assay Method Information | |
| | Competition Binding Assay |
| Description: | Competition binding with α2AR subtypes were performed by incubating membranes in buffer A (50 mM TRIS at pH 7.4) at final protein concentrations of 3-10 g/well with the radioligand (final concentration 0.5-2.0 nM according to the appropriate KD and Bmax) and varying concentrations of the competing ligands for 60 minutes at 37° C. Binding to α1A and α1B was measured with buffer B (50 mM TRIS, 5 mM MgCl2, 1 mM EDTA, 100 μg/mL bacitracin and 5 μg/mL soybean trypsin inhibitor at pH 7.4) at 2-6 g/well (radioligand at 0.2-0.3 nM) and binding to 1 and 12 was measured with buffer C (25 mM HEPES, 5 mM MgCl2, 1 mM EDTA, and 0.006% bovine serum albumin at pH 7.4) at 4-8 μg/well (radioligand 0.2 nM). Non-specific binding was determined in the presence of unlabeled ligand at 10 μM. Protein concentration was measured using the method of Lowry (71). |
| Affinity data for this assay | |
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