| Assay Method Information | |
| | Competition Binding Assay |
| Description: | The protein concentration of membrane suspension was determined using the Bradford method (Pierce, Rockford, Ill., USA) with bovine albumin as standard. Competition binding experiments were performed by incubating membranes (5-10 μg of protein/sample) with a single concentration of the A2a antagonist [3H]ZM241385 (Biotrend, Cologne, Germany) (2 nmol/l), in the presence of various concentrations (ranging from 10−5 to 10−11 mol/l) of test and reference compounds in 96-well filter plates (MultiScreen system, cat. MAFBN0B10, Millipore, Billerica, Mass., USA) for one h at 4° C. in a total volume of 200 μl/well of appropriate buffer (50 mmol/l Tris-HCl, pH 7.4, 10 mmol/l MgCl2). Nonspecific binding was determined in the presence of 10 μmol/l cold ZM241385 (Tocris, Ellisville, Mo., USA). At the end of incubation, bound and free radioligands were separated by filtering the 96-well filter plates using a Millipore filtration apparatus (MultiScreenHTS vacuum manifold). |
| Affinity data for this assay | |
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