| Assay Method Information | |
| | Radiological Binding Assay |
| Description: | For A1 adenosine receptors, 40 mg of protein were incubated for 60 min at 25°C with [3H]DPCPX 0.5 nM (Kd = 0.4 nM) and increasing concentrations of the compounds, in a final volume of 0.4 mL of Tris-HCl buffer. Binding reaction was terminated by dilution with ice-cold 50 mM Tris-HCl buffer, pH 7.4.For A2A and A3 adenosine receptors, 40 mg of protein were incubated with [3H]ZM241385 6 nM (Kd = 4 nM) in the experiments involving A2A and with [3H]NECA 15 nM (Kd = 150 nM) involving A3 and the compounds to be assayed, at fixed concentration (10 µM) or at increasing concentrations of the compounds in duplicate, in a final volume of 0.4 mL of Tris-HCl buffer for 120 min at 25°C. Non-specific binding was measured in the presence of 100 µM NECA in the case of A2A and 100 µM R-PIA in the case of A3 binding assay. Bound radioactivity of all samples was measured in a liquid scintillation counter (1600 TR Packard) after the addition of 4 mL of scintillation liquid (Emulsifier-Safe; Hewlett-Packa |
| Affinity data for this assay | |
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