PDE3A Assay Kit
Catalog # 60330
Size: 96 reactions
DESCRIPTION: 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. The PDE3A
Assay Kit is designed for identification of PDE3A inhibitors using fluorescence
polarization. The assay is based on the binding of a fluorescent nucleotide
monophosphate generated by PDE3A 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.
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.
APPLICATIONS: Great for studying enzyme kinetics and screening small molecular
inhibitors for drug discovery and HTS applications.
REFERENCE: Maurice DH. Front. Biosci. 2005; 10:1221-8.