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Assay Method Information

Assay Name:  Inhibition Assay
Description:  Inhibition of the human PDE4 enzyme, using semi-purified enzyme from human U937 cells. The PDE4 enzyme was partially purified from human U937 myeloid leukemia cells and activity was assayed using a method optimized from Cortijo et al (10, Cortijo 1993) and Nicholson et al. (11, Nicholson, 1991). Test article and/or vehicle was incubated with 0.2 mg of enzyme and 1 μM cAMP containing 0.01 μM [3H]cAMP in Tris buffer (50 mM Tris-HCl pH 7.5, 5 mM MgCl2) for 20 minutes at 25° C. The reaction was terminated by boiling for 2 minutes and the resulting AMP was converted to adenosine by addition of 10 mg/ml snake venom nucleotidase and further incubation at 37° C. for 10 min. Unhydrolyzed cAMP was bound to AG1-X2 resin, and the remaining [3H] adenosine in the aqueous phase was quantitated by scintillation counting.
Affinity data for this assay
 

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Last update November 1, 2007
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