| Assay Method Information | |
| | Adenosine Receptor Binding Assay |
| Description: | Inhibition binding assays were performed using 0.2 µg of membranes prepared from HEK293 cells infected with BacMam human adenosine A2A receptor or 1.4 µg of membranes prepared from HEK293 cells infected with BacMam human adenosine A1 receptor. Membranes were incubated in 50 mM Tris-HCl (HEK293-hA2A; pH 7.4) or 50 mM Tris-HCl, 100 mM NaCl, 10 mM MgCl2 (CHO-hA1; pH 7.4) in the presence of varying concentrations of test compound and 1 nM [3H]ZM241385 (HEK293-hA2A) or [3H]DPCPX (CHO-hA1) at 25° C. for 1 h. The assay was then terminated by rapid filtration onto GF/B grade Unifilter plates using a TomTec cell harvester, followed by 5 × 0.5 ml washes with ddH2O. Nonspecific binding was defined in the presence of 1 µM CGS 15943 (HEK293-hA2A) or 1 µM DPCPX (CHO-hA1). Bound radioactivity was determined by liquid scintillation counting and inhibition curves were analysed using a four-parameter logistic equation. IC50 values were converted to Ki values with the Cheng-Prusoff equation using a KD value derived from saturation binding studies. |
| Affinity data for this assay | |
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