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. Increased expression of PDE4B2 was observed in the near-term myometrium. PDE4B2 can be induced by its own substrate, under the control of one of the major utero-contractile agonist, PGE2.
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 PDE4B2 Assay Kit is designed for identification of PDE4B2 inhibitors using fluorescence polarization. The assay is based on the binding of a fluorescent nucleotide monophosphate generated by PDE4B2 to the binding agent.
Test Priniciple:The PDE4B2 inhibitor screening assay kit comes in a convenient 96-well format, with purified PDE4B2 enzyme, fluorescently labeled PDE3 substrate (cAMP), binding agent, and PDE assay buffer for 100 enzyme reactions. The key to the PDE4B2 Assay Kit is the specific binding agent. Using this kit, only two simple steps on a microtiter plate are required for PDE4B2 reactions. First, the fluorescently labeled cAMP is incubated with a sample containing PDE4B2 for 1 hour. Second, 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.
Storage and Stability:Store *137341D at -20ºC. Store *137341B and *137341C at -70ºC. Store all other components at 4°C. Stable for 6 months after receipt. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
