Phosphodiesterases (PDEs) play an important role in dynamic regulation of cAMP and cGMP signaling. PDE3A, also known as cGMP-inhibited phosphodiesterase, has been implicated in cardiovascular function and fertility.
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.
The PDE3A inhibitor screening assay kit comes in a convenient 96-well format, with purified human PDE3A enzyme, fluorescently labeled PDE3 substrate (cAMP), binding agent, and PDE assay buffer for 100 enzyme reactions. The key to the PDE3A Assay Kit is the specific binding agent. Using this kit, only two simple steps on a microtiter plate are required for PDE3A reactions. First, the fluorescently labeled cAMP is incubated with a sample containing PDE3A 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.
The PDE3A inhibitor screening assay kit comes in a convenient 96-well format, with purified PDE3A enzyme, fluorescently labeled PDE3 substrate (cAMP), binding agent, and PDE assay buffer for 100 enzyme reactions. The key to the PDE3A Assay Kit is the specific binding agent. Using this kit, only two simple steps on a microtiter plate are required for PDE3A reactions. First, the fluorescently labeled cAMP is incubated with a sample containing PDE3A 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 *506363D at -20ºC. Store *506363B and *506363C at -70ºC. Store all other components at 4°C. Unused 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 polarizationAdjustable micropipettor and sterile tips1,4-Dithiothreitol (DTT), 1M in anhydrous DMSO
Applications:Great for studying enzyme kinetics and screening small molecular inhibitors for drug discovery and HTS applications.
