| Assay Method Information | |
| | Scintillation Proximity Assay (SPA) |
| Description: | The inhibition of recombinant human (rh) PDE5A by test compounds is measured in a radiometric assay based on Scintillation Proximity Assay (SPA) technology. The substrate [3H]cGMP/cGMP is hydrolysed to [3H] 5′ GMP/5′ GMP contingent on the activity of rhPDE5A. The ensuing [3H] 5′ GMP/5′ GMP but not [3H] cGMP/cGMP binds to SPA yttrium silicate beads in the presence of Zn++ stimulating the scintillant within the bead to emit light that is detected by a -counter. The assay is performed in a 96 well format.The assay is done in 20 mM Tris HCl pH 7.4, 5 mM MgCl2, 0. μM cGMP/[3H] cGMP (about 60000 dpm/well) substrate with rhPDE5A1 (GST tagged, SIGMA E9034) added to an amount not exceeding 20% cGMP hydrolysis within 20 min in Tris 20 mM pH 7.4 supplemented with 0.01% bovine serum albumin (BSA) in the presence of test compounds or vehicle (0.1% DMSO). The final assay volume amounts to 100 μl and the reaction is run for 20 min at 37° C.The hydrolysis of [3H] cGMP/cGMP by rhPDE5A is terminated by adding SPA beads at 50 l/well (Perkin Elmer, RPNQ0024), pre-diluted in water as per manufacturer's instructions and supplemented with 3-isobutyl-1-methylxanthine (1 mM). Beads are allowed to sediment for at least 30 min before measurement in a Wallac Microbeta 2 (Perkin Elmer). |
| Affinity data for this assay | |
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