| Assay Method Information | |
| | PDE10A assay |
| Description: | Human PDE10A IC50 values were determined by using purified enzyme (Biomol cat. # SE-534) in a scintillation proximity assay (SPA) in 96 well plates (OptiPlate white, Perkin Elmer) and using Yttrium silicate microsphere beads (Y-Si beads, Perkin Elmer). Compounds were diluted in 20 mM HEPES/NaOH (pH 7.5), 10 mM MgCl2 and 0.05 mg/ml bovine serum albumin. A fixed amount of 2 ng/well human PDE10A enzyme was added and incubated at room temperature for 15 min under agitation. The reaction was started by the addition of the substrate cocktail [3H]cGMP (Perkin Elmer NET337001MC, S.A. 13.5 Ci/mmol) / cGMP at a final concentration of 50 nM and incubated for an additional 15 min. The reaction was stopped by the addition of 0.5 mg/well of Y-Si SPA beads. The plates were shaken for 1 h, followed by a brief centrifugation (170 g for 1 min). The radioactivity bound to the beads was estimated using a TopCount (Perkin-Elmer). For the determination of IC50 values, a four parameters logistics equation was used. pIC50 is defined as − log10(IC50). |
| Affinity data for this assay | |
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