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Applications: Great for studying enzyme kinetics and screening small molecular inhibitors for drug discovery and HTS applications.
Background: Phosphodiesterases (PDEs) play an important role in the dynamic regulation of cAMP and cGMP signaling. PDE1A1 is a calmodulin-dependent PDE and expressed in lung and heart. PDE1A1 is believed to regulate vascular smooth muscle contraction and may play a role in sperm function. Phosphodiesterases catalyze the hydrolysis of the phosphodiester bond in dye-labeledcyclic monophosphates. Beads selectively bind the phosphate group in the nucleotideproduct. This increases the size of the nucleotide relative to unreacted cyclicmonophosphate. In the polarization assay, dye molecules with absorption transitionvectors parallel to the linearly-polarized excitation light are selectively excited. Dyesattached to the rapidly-rotating cyclic monophosphates will obtain random orientationsand emit light with low polarization. Dyes attached to the slowly-rotating nucleotide-beadcomplexes will not have time to reorient and therefore will emit highly polarized light.
Contraindications: DMSO >1%, strong acids or bases, ionic detergents, high salt
Description: The PDE1A Assay Kit is designed for identification of PDE1A inhibitors using fluorescence polarization. The assay is based on the binding of a fluorescent nucleotide monophosphate generated by PDE1A1 to the binding agent. The key to the PDE1A Assay Kit is the specific binding agent. Using this kit, only two simple steps on a microtiter plate are required for PDE1A reactions. First, the fluorescently labeled cAMP is incubated with a sample containing PDE1A1 for 1 hour. Second, a binding agent is added to the reaction mix to produce a change in fluorescent polarization. The FP signal is measured using a fluorescent microplate reader capable of measuring fluorescence polarization.
Storage Stability: At least 6 months from date of receipt, when stored as directed. Kit components require different storage conditions. Be sure to store each component at the proper temperature upon arrival.
Supplied As: The PDE1A inhibitor screening assay kit comes in a convenient 96-well format, including purified PDE1A1 enzyme, fluorescently labeled PDE1A substrate (cAMP), binding agent, and PDE assay buffer for 100 enzyme reactions.
Warnings: Avoid freeze/thaw cycles.
Biosafety Level: Not applicable (BSL-1)
References: Bender. A., et al. Pharmacol. Rev. 2006; 58: 488-520.
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