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 PDE5A1Assay 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.
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.
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
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.
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.
Instructions for Use
See datasheet for detailed protocol.
Applications
Useful for studying enzyme kinetics and screening small molecular inhibitors for drug discovery and HTS applications.
Shipping Temperature
-80°C
Warnings
Avoid freeze/thaw cycles
Disclaimers and Limitations
BPS Bioscience assay kits are validated using the listed components according to our specific protocol. Any deviations to kit components or protocols, such as using different proteins, cell/tissue lysates, or buffers (including using the same component from other commercial sources) will void any warranties on the performance of the assay kit and is not recommended.
End-users should immediately report any issues to [email protected] upon receipt of the kit, such as missing components, damaged vials, no dry ice, etc.