| Assay Method Information | |
| | Evaluation of A2a Receptor Binding Affinity |
| Description: | A radioligand binding test was conducted by using [3H]-NECA (5′-N-[adenine-2,8-3H]-ethylcarboxamidoadenosine) and an adenosine A2a membrane. As the adenosine A2a membrane, a cell membrane prepared from HEK-293 cells transfected with human adenosine A2a receptor was used. The membrane used for the test was prepared by incubating with a radioligand until equilibrium was reached. In order to separate the radioligand-bound membrane, the unbound radioligand was separated by using Packard filtermate Harverster and glass filter plates.10 μL of test compound dissolved in binding buffer (50 mM Tris, 10 mM MgCl2, 1 mM EDTA pH 7.4) and 20 μL of [3H]-NECA (final concentration of 37 nM) or reference inhibitor was mixed with 20 μL of A2a membrane in an unbound 96-well plate and incubated at room temperature for one hour. Prior to filtration, a 96-well harvest filter plate was coated with 0.33% polyethylenimine for 30 minutes and then washed with assay buffer. The binding reaction was transferred to the filter plate and washed three times with wash buffer. The dish was then dried, scintillant was added, and radioactivity was counted in a scintillation counter (Topcount NXT, Packard). |
| Affinity data for this assay | |
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