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| | Radioligand Competition Binding Assay |
| Description: | For competition binding experiments, 0.5 mg of the rat brain homogenates in 800 μL of binding buffer (2 mM MgCl2 in 50 mM Tris*HCl, pH=7.4) were incubated on a thermoshaker at 37° C. for 30 min with 100 μL (R)-11C-3 or (R)-18F-11 and 100 μL of the compound over a range of concentrations (from 0.1 to 100 nM). Nonspecific binding was defined as the residual binding observed in the presence of 1 mM levetiracetam (100 μL). At completion of the incubation period, the membrane-bound radioligand was recovered using rapid filtration through GF/B glass fiber filters (13 mm GD/X, Whatman Inc.) which were pre-soaked for at least 30 min in 0.5% polyethyleneimine (PEI). The membranes were washed twice with 1 mL of ice-cooled binding buffer. The binding mixtures, filters and filtrates were counted using cross-calibrated gamma counters (1480 & 2480 WIZARD; Perkin-Elmer). Values of IC50 were calculated using the GRAPHPAD PRISM software and converted to inhibition constants (Ki) by the Cheng-Prusoff equation. |
| Affinity data for this assay | |
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