| Assay Method Information | |
| | Inhibition of Recombinant PDE2A |
| Description: | The DNA encoding PDE2A (NM002599) was cloned and the gene was inserted in the baculovirus and the enzyme-protein expressed in SF21-cells. The enzyme was isolated from these cells by harvesting the cells by an centrifugation at 200 g to collect the cells. The cells were resuspended in 50 mM Tris-HCl/5 mM MgCl2 buffer (pH=7.4)(Sigma, Deisenhofen, Germany; Merck, Darmstadt, Germany) and lysed by a sonication of the cells (three times for 15 seconds, Labsonic U, Fa. Braun, Degersheim, Switzerland, level high). The membrane fraction of PDE2A was obtained by a centrifugation at 48 000 g for 1 h, resuspended in buffer and stored at −70° C.PDE2A activity was determined in a one step procedure in microtiterplates. The reaction mixture of 100 μl contained 50 mM Tris-HCl/5 mM MgCl2 buffer (pH=7.4)(Sigma, Deisenhofen, Germany; Merck, Darmstadt, Germany), 0.5 OA [3H]-cAMP (PerkinElmer, Shelton, USA), 1000 nM cGMP and the enzyme. Non-specific enzyme activity was tested in the absence of cGMP. The reaction was initiated by addition of the substrate solution and was carried out at 37° C. for 30 minutes. Enzymatic activity then was stopped by addition of 25 μl SPA-beads (PerkinElmer, Shelton, USA). One hour later the mixture was measured in a liquid scintillation counter for microtiterplates (Microbeta Trilux). For pipetting of the incubation mixture we routinely use the robot Biomek (Fa. Beckman). |
| Affinity data for this assay | |
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