Phosphodiesterase’s (PDEs) play an important role in the dynamic regulation of cAMP and cAMP signaling. Rat PDE4D, also known as cAMP-inhibited phosphodiesterase, has been implicated in cardiovascular function and fertility.
The Rat PDE4D Assay Kit is designed for identification of inhibitors of Rat PDE4D using fluorescence polarization. The assay is based on the binding of a fluorescent nucleotide monophosphate generated by Rat PDE4D 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 key to the Rat PDE4D Assay Kit is the specific binding agent. Using this kit, only two simple steps on a microtiter plate are required for Rat PDE4D reactions. First, the fluorescently labeled cAMP is incubated with a sample containing Rat PDE4D 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.
