| Assay Method Information | |
| | Adenosine Receptor Binding Assay |
| Description: | Radioligand-binding assays were performed using the adenosine A3 receptor agonist [125I]-AB-MECA and the A1 receptor agonist [3H]CCPA. Binding assays were conducted in a total volume of 100 μL containing a 50 mM Tris, pH 7.4 buffer with or without 10 mM MgCl2, 20 μg membranes and either 0.15 nM [125I]-AB-MECA (for the A3 receptor) or 1 nM [3H]CCPA (for the A1 receptor). Assays were conducted at room temperature for 60 min (A1 receptor) or 120 min (A3 receptor) and terminated by the addition of 2 ml of ice-cold wash buffer (50 mM Tris, pH 7.4 with or without 10 mM MgCl2) and rapid filtration over 0.03% polyethylenimine-treated Whatman GF/C filters using a Brandel cell harvester (Semat International Ltd., St Albans, UK). Filters were then washed three times with 2 ml of ice-cold buffer. The filter-bound radioactivity were counted using a Compugamma counter (LKB Wallac, Turku, Finland). Competition experiments were performed to investigate the ability of reference compounds and embodiments of the invention to inhibit [125I]-AB-MECA binding. Non-specific binding were determined using the respective compound at its highest concentration |
| Affinity data for this assay | |
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