PDE6C Assay Kit

The PDE6C Assay Kit is designed for identification of PDE6C inhibitors using fluorescence polarization. The assay is based on the binding of a fluorescent nucleotide monophosphate generated by PDE6C 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 PDE6C inhibitor screening assay kit comes in a convenient 96-well format, with purified PDE6C enzyme, fluorescently labeled PDE6 substrate (cGMP), binding agent, and PDE assay buffer for 100 enzyme reactions. The key to the PDE6C Assay Kit is the specific binding agent. Using this kit, only two simple steps on a microtiter plate are required for PDE6C reactions. First, the fluorescently labeled cGMP is incubated with PDE6C for 1 hour. Second, the 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.