Phosphodiesterases (PDEs) play an important role in dynamic regulation of cAMP and cGMP signaling. PDE8 is involved in many biological processes, including T-cell activation, testosterone production, adrenocortical hyperplasia, and thyroid function.
Intended Use:The PDE8A Assay Kit is designed for identification of PDE8A inhibitors using fluorescence polarization. The assay is based on the binding of a fluorescent nucleotide monophosphate generated by PDE8A 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.
Test Principle:The PDE8A inhibitor screening assay kit comes in a convenient 96-well format, including purified PDE8A enzyme, fluorescently labeled PDE8A substrate (cAMP), binding agent, and PDE assay buffer for 100 enzyme reactions. The key to the PDE8A Assay Kit is the specific binding agent. Using this kit, only two simple steps on a microtiter plate are required for PDE8A reactions. First, the fluorescently labeled cAMP is incubated with a sample containing PDE8A 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.
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.
Storage and Stability:Store other components at 4°C. Stable for 6 months. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
