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Assay Method Information

Assay Name:  Radioligand Binding Assay
Description:  Dissociation constants at A1, A2A and A3 receptors (Ki-values) were determined in radioligand competition experiments as reported earlier [19,20]. All binding experiments were done in a microplate format utilizing a 96-well microplate filtration system (Millipore Multiscreen MAFC). As radioligands the agonists [3H]CCPA (1 nM) for A1, [3H]NECA (10 nM) for A2A, and [3H]HEMADO (1 nM) for A3 receptors were used. Samples were incubated with 10 g of membrane protein for 3 h at 25 C, filtered through the built-in filter at the bottom of the wells and washed three times with 200 ml of ice-cold binding buffer. After addition of 20 l of scintillator to the dried filter plates samples were counted in a Wallac Micro-Beta counter. Nonspecific binding was determined in the presence of 1 mM theophylline ([3H]CCPA) or 100 mM R-PIA (N6-phenylisopropyladenosine; [3H]NECA and [3H] HEMADO).
Affinity data for this assay
 

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Last update November 1, 2007
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