PDE4B3 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. PDE4B3 mRNA is readily expressed in both oligodendrocytes and neurons and it is the first cAMP-specific phosphodiesterase to be associated with hippocampal long-term potentiation (LTP), the most prominent cellular model for learning and memory formation. The PDE4B3 Assay Kit is designed for identification of inhibitors of PDE4B3 using fluorescence polarization. The assay is based on the binding of a fluorescent nucleotide monophosphate generated by PDE4B3 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 PDE4B3 Assay Kit comes in a convenient 96-well format, with purified PDE4B3 enzyme, fluorescently labeled PDE4B3 substrate (cAMP), binding agent, and PDE assay buffer for 100 enzyme reactions. The key to the PDE4B3 Assay Kit is the specific binding agent. Using this kit, only two simple steps on a microtiter plate are required for PDE4B3 reactions. First, the fluorescently labeled cAMP is incubated with a sample containing PDE4B3 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.