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Assay Method Information

Assay Name:  Inhibition Assay
Description:  The test substances are dissolved in 100% DMSO and serially diluted to determine their in vitro effect on PDE 9A. Typically, serial dilutions from 200 uM to 1.6 uM are prepared (resulting final concentrations in the assay: 4 uM to 0.032 uM). 2 uL portions of the diluted substance solutions are introduced into the wells of microtiter plates (Isoplate; Wallac Inc., Atlanta, Ga.). Then 50 uL of a dilution of the PDE9A preparation described above are added. The dilution of the PDE9A preparation is chosen so that less than 70% of the substrate is converted during the subsequent incubation (typical dilution: 1:10000; dilution buffer: 50 mM Tris/HCl pH 7.5, 8.3 mM MgCl2, 1.7 mM EDTA, 0.2% BSA). The substrate, [8-3H] guanosine 3'-cyclic phosphate (1 uL; Amersham Pharmacia Biotech., Piscataway, N.J.) is diluted 1:2000 with assay buffer (50 mM Tris/HCl pH 7.5, 8.3 mM MgCl2, 1.7 mM EDTA) to a concentration of 0.0005 uL.
Affinity data for this assay
 

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Last update November 1, 2007
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