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Phosphodiesterases (PDEs) play an important role in dynamic regulation of cAMP and cGMP signaling. PDE9A2 is primarily expressed in spleen, small intestine, and brain. PDE9 inhibitors have been studied as therapeutics for treatment of cardiovascular diseases, diabetes, and neurodegenerative disorders. The PDE9A2 Assay Kit is designed for identification of PDE9A2 inhibitors using fluorescence polarization. The assay is based on the binding of a fluorescent nucleotide monophosphate generated by PDE9A2 to the binding agent.
Phosphodiesterases catalyze the hydrolysis of the phosphodiester bond in dye-labeled cyclic monophosphates. Beads selectively bind the phosphate group in the nucleotide product. This increases the size of the nucleotide relative to unreacted cyclic monophosphate. In the polarization assay, dye molecules with absorption transition vectors parallel to the linearlypolarized excitation light are selectively excited. Dyes attached to the rapidly-rotating cyclic monophosphates will obtain random orientations and emit light with low polarization. Dyes attached to the slowly-rotating nucleotide-bead complexes will not have time to reorient and therefore will emit highly polarized light. The Fluorescence Polarization signal is measured using a fluorescent microplate reader capable of measuring fluorescence polarization.
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