PDE5A1 Assay Kit

Catalog #
60350
$455 *
Size: 96 reactions
Qty
*US Pricing only. For international pricing, please contact your local distributor.
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Description

Phosphodiesterases (PDEs) play an important role in dynamic regulation of cAMP and cGMP signaling. PDE5A1, also known as cGMP-inhibited phosphodiesterase, has been implicated in cardiovascular function and fertility. The PDE5A1 Assay Kit is designed for identification of PDE5A1 inhibitors using fluorescence polarization. The assay is based on the binding of a fluorescent nucleotide monophosphate generated by PDE5A1 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.

PDE5A Assay Kit

The key to the PDE5A1 Assay Kit is the specific binding agent. Using this kit, only two simple steps on a microtiter plate are required for PDE5A1 reactions. First, the fluorescently labeled cGMP is incubated with PDE5A1 for 1 hour. Second, the 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.

Need us to run inhibitor screens or profile your compounds against PDE5A1? Check out our Phosphodiesterase Screening Services.

This product has been cited 13 times.

Synonyms
cGMP-specific 3',5'-cyclic phosphodiesterase, cGMP-binding cGMP-specific phosphodiesterase, CGB-PDE, PDE5A, PDE5, PDE5-A1, PDE
Product Info
Storage and Usage
Citations13
Assay Kit Format
Fluorescence Polarization
Supplied As
The PDE5A1 inhibitor screening assay kit comes in a convenient 96-well format, with purified PDE5A1 enzyme, fluorescently labeled PDE5 substrate (cGMP), binding agent, and PDE assay buffer for 100 enzyme reactions.
Materials Required But Not Supplied
Fluorescent microplate reader capable to measure fluorescence polarization
Format
Catalog # Component Amount Storage
60050 PDE5A1 >1 µg -80°C Avoid freeze/ thaw cycles
60201 FAM-Cyclic-3′, 5′-GMP: 20 µM 50 µl -80°C
60393 PDE assay buffer 25 ml -20°C
60390 Binding Agent 100 µl +4°C
60392 Binding Agent Diluent (cGMP) 10 ml +4°C
79685 Black, low binding, microtiter plate 1 Room temp.
UniProt #
O76074
Background
Phosphodiesterases (PDEs) play an important role in the dynamic regulation of cAMP and cGMP signaling. PDE5A1, 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.
References

Maurice DH. Front. Biosci. 2005; 10:1221-8.