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| | Assay for Binding Affinity for Adenosine Receptor A1 |
| Description: | CHO cell membrane homogenates (40 μg protein), in which A1 adenosine receptors were expressed, are incubated for 60 min at 22° C. with 1 nM [3H]CCPA in the absence or presence of the test compound in a buffer containing 50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 1 mM EDTA, 2 UI/ml ADA, 1 g/ml Leupeptin, 1 M Pepstatin and 10 μg/ml Trypsin inhibitor. Nonspecific binding is determined in the presence of 10 μM CPA. Following incubation, the derivatives according to embodiments of the present disclosure are filtered rapidly under vacuum through glass fiber filters (GF/B, Packard) presoaked with 0.3% PEI and rinsed several times with ice-cold 50 mM Tris-HCl using a 96-sample cell harvester (Unifilter, Packard). The filters are dried then counted for radioactivity in a scintillation counter (Topcount, Packard) using a scintillation cocktail (Microscint 0, Packard). The results are expressed as a percent inhibition of the control radioligand specific binding. The standard reference compound is CPA, which is tested in each experiment at several concentrations to obtain a competition curve from which its IC50 is calculated. |
| Affinity data for this assay | |
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