The PDE7A Assay Kit is designed for identification of PDE7A inhibitors using fluorescence polarization. The assay is based on the binding of a fluorescent nucleotide monophosphate generated by PDE7A to the binding agent. PDE7A catalyzes the hydrolysis of the phosphodiester bond in dye-labeled cyclic adenosine monophosphate (cAMP). Nanoparticle beads selectively bind the phosphate group in the nucleotide product. This increases the size of the nucleotide relative to unreacted cAMP. Since the degree of polarization of a fluorophore is inversely related to its molecular rotation, dyes attached to the slowly-rotating nucleotide-bead complexes will not have time to reorient and therefore will emit highly polarized light. Conversely, dyes attached to the rapidly-rotating cyclic monophosphates will obtain random orientations and emit light with low polarization. The key to the PDE7A Assay Kit is the specific binding agent. Using this kit, only two simple steps on a microtiter plate are required for PDE7A reactions. First, the fluorescently labeled cAMP is incubated with a sample containing PDE7A for 1 hour. Second, a binding agent is added to the reaction mix to produce a change in fluorescent polarization that can then be measured using a fluorescence reader.
