PDE4B (Rat) Assay Kit

Phosphodiesterases (PDEs) play an important role in dynamic regulation of cAMP and cGMP signaling. PDE4 selective inhibitors are currently in clinical trials for the treatment of diseases related to inflammatory disorders. Altered activity of PDE4B has been associated with schizophrenia and bipolar disorder. It has also recently been found to modulate cognition, as reduction in PDE4B activity improves memory and long-term plasticity in mouse models. The Rat PDE4B Assay Kit is designed for identification of inhibitors of rat PDE4B (rPDE4B) using fluorescence polarization. The assay is based on the binding of a fluorescent nucleotide monophosphate generated by rPDE4B 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 linearly-polarized 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 Rat PDE4B Assay Kit comes in a convenient 96-well format, with purified rPDE4B enzyme, fluorescently labeled rPDE4B substrate (cAMP), binding agent, and PDE assay buffer for 100 enzyme reactions. The key to the Rat PDE4B Assay Kit is the specific binding agent. Using this kit, only two simple steps on a microtiter plate are required for rPDE4B reactions. First, the fluorescently labeled cAMP is incubated with a sample containing rPDE4B 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 equipped for the measurement of fluorescence polarization.

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