| Assay Method Information | |
| | Beta-2 AR binding assay |
| Description: | HEK 293 cells stability transfected with cDNA encoding human β2-AR (provided by Dr. Brian Kobilka, Stanford Medical Center, Palo Alto, Calif.) were grown in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS), 0.05% penicillin-streptomycin, and 400 μg/ml G418 as previously described (Pauwels et al., Biochem. Pharmacol. 42: 1683-1689, 1991). The cells were scraped from the 150×25 mm plates and centrifuged at 500×g for 5 minutes. The pellet was homogenized in 50 mM Tris-HCl, pH 7.7, with a Polytron, centrifuged at 27,000×g, and resuspended in the same buffer. The latter process was repeated, and the pellet was resuspended in 25 mM Tris-HCl containing 120 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgCl2, and 5 mM glucose, pH 7.4. The binding assays contained 0.3 nM [3H]CGP-12177 in a volume of 1.0 ml. Nonspecific binding was determined by 1 μM propranolol.According to the above-described methods, binding affinities, expressed as Ki values, were determined using membranes obtained from a HEK 293 cell line stably transfected with cDNA encoding human β2-AR (Pauwels et al., Biochem. Pharmacol. 42: 1683-1689, 1991) with [3H]CGP-12177 as the marker ligand. The resulting IC50 values and Hill coefficients were calculated for each test compound using GraphPad Prism software and Ki values were calculated using the Cheng-Prusoff transformation (Biochem Pharmacol 22: 3099-3108, 1973):K i=IC50/(1+L/K d)+ Eqn. 1.Where: L is the concentration of [3H]CGP-12177 and Kd is the binding affinity of the [3H]CGP-12177. Each test compounds was assayed three times. |
| Affinity data for this assay | |
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