Phosphodiesterases (PDEs) play an important role in dynamic regulation of cAMP and cGMP signaling. PDE4A is widely expressed in brain tumors and promotes their growth and its inhibitor Rolipram is identified as a potent anti- inflammatory agent. The PDE4A Assay Kit is designed for identification of PDE4A inhibitors using fluorescence polarization. The assay is based on the binding of a fluorescent nucleotide monophosphate generated by PDE4A to the binding agent.
Intended Use:Designed for identification of PDE4A inhibitors using fluorescence polarization.
Test Principle:The PDE4A1A Assay Kit is designed for identification of PDE4A1A inhibitors using fluorescence polarization. The assay is based on the binding of a fluorescent nucleotide monophosphate generated by PDE4A1A 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. For illustration of these processes, please see Fig. 1 below.
The PDE4A1A inhibitor screening assay kit comes in a convenient 96-well format, including purified PDE4A1A enzyme, fluorescently labeled PDE4A substrate (cAMP), binding agent, and PDE assay buffer for 100 enzyme reactions. The key to the PDE4A1A Assay Kit is the specific binding agent. Using this kit, only two simple steps on a microtiter plate are required for PDE4A1A reactions. First, the fluorescently labeled cAMP is incubated with a sample containing PDE4A1A 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.
Storage and Stability:Store 137340D* at -20ºC. Store 137340B* and 137340C* at -70ºC. Store all other components at 4°C. Unopened kit is stable for 6 months after receipt. For maximum recovery of product contained in small vials, pulse spin after thawing and prior to removing the cap.
Materials or Instruments Required But Not Supplied:Fluorescent microplate reader capable of measuring fluorescence polarization
Applications:Great for studying enzyme kinetics and screening small molecular inhibitors for drug discovery and HTS applications.
Contraindications:DMSO >1%Strong acids or basesIonic detergentsHigh salt
