| Assay Method Information | |
| | Radioligand Binding Assay |
| Description: | For all four adenosine receptors (A1, A2A, A2B and A3) filtration binding assay was performed. Radioligand binding competition assay was done in duplicates in the wells of a 96-well plate (Master Block, Greiner, 786201) containing binding buffer, receptor membrane extracts, a fixed concentration of tracer and test compound at increasing concentrations. In order to eliminate effect of buffer components, binding buffer was the same for all four receptors and contained: 50 mM Tris-HCl pH 7.4, 5 mM MgCl2, 1 mM EDTA, 150 mM NaCl, 0.1% Na-azide, and 5 U/ml adenosine-deaminase. Nonspecific binding was determined by co-incubation with 200-fold excess of cold competitor. In all radioligand binding experiments, the samples were incubated in a final volume of 0.1 ml for 60 minutes at 25° C. and then filtered over Unifilter plates (Perkin Elmer) pre-treated for 2 hours to limit tracer non-specific binding. Filters were washed five times with 0.5 ml of ice-cold washing buffer (50 mM Tris-HCl pH 7.4, 5 mM MgCl2, 1 mM EDTA) and 50 μl of Microscint 20 (Perkin Elmer) were added to each filter. The plates are incubated 15 min at room temperature on an orbital shaker and then counted with a TopCount™ for 1 min/well. |
| Affinity data for this assay | |
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