The PDE3B Assay Kit is designed for identification of PDE3B inhibitors using fluorescence polarization. The assay is based on the binding of a fluorescent nucleotide monophosphate generated by PDE3B to the binding agent. The key to the PDE3B Assay Kit is the specific binding agent. Using this kit, only two simple steps on a microtiter plate are required for PDE3B reactions. First, the fluorescently labeled cAMP is incubated with a sample containing PDE3B 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 PDE3B inhibitor screening assay kit comes in a convenient 96-well format, withpurified PDE3B enzyme, fluorescently labeled PDE3 substrate (cAMP), binding agent,and PDE assay buffer for 100 enzyme reactions.
Format
UniProt #
Q14432
Background
Phosphodiesterases (PDEs) play an important role in dynamic
regulation of cAMP and cGMP signaling. PDE3B, also known as cGMP-inhibited
phosphodiesterase, is involved in mediating the anti-lipolytic and anti-glycogenolytic effects of
insulin in adipose and liver tissues. 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 assay kit data sheet for detailed protocol.
Applications
Useful for studying enzyme kinetics and screening small molecular inhibitors for drug discovery and HTS applications.
Shipping Temperature
-80°C
Notes
MATERIALS OR INSTRUMENTS REQUIRED BUT NOT SUPPLIED:
Fluorescent microplate reader capable to measure fluorescence polarization
Contraindications
DMSO >1%, strong acids or bases, ionic detergents, high salt
Warnings
This assay requires a fluorescent microplate reader capable of measuring fluorescence polarization (FP) and equipped with the required partsto read the FP signal. For more information FP technology, visit our Tech Note: FP, assay principles and applications.
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.
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