PDE8A Assay Kit

• Background:

  Phosphodiesterases (PDEs) play an important role in the dynamic regulation of the second messengers cAMP (cyclic adenosine monophosphate) and cGMP (cyclic guanosine monophosphate) signaling, by hydrolyzing them. The PDE superfamily is composed of 11 families, with PDE4,7 and 8 being cAMP-specific hydrolases, and thus regulating positive and negative responses to it. PDE8 enzymes, encoded by the PDE8A and PDE8B genes, display high affinity and specificity for cAMP. Studies using PDE8A and PDE8B knock-out mice have identified important functions of the PDE8 family in steroidogenesis. Leydig cells from PDE8A knock-out mice show increased sensitivity to lutenizing hormone for testosterone production. PDE8A is also important in other processes such as T cell activation, effector T cell adhesion and excitation-contraction coupling in ventricular myocytes.

• Description:

  The PDE8A1 Assay Kit is a fluorescence polarization (FP), homogeneous, 96-well assay kit designed for the screening and profiling of PDE8A1 (Phosphodiesterase 8A1) inhibitors. This assay takes advantage of a specific fluorescent phosphate-binding nanoparticle. The kit contains enough purified recombinant PDE8A1, fluorescent probe, PDE assay buffer, Binding Agent, and diluent for 100 reactions.Figure 1: Illustrationof the PDE8A1 Assay Kit principle.The assay uses a fluorescein-labeled cyclic adenosine monophosphate (cAMP-FAM for PDE8A1), in which the phosphate group is engaged within the cyclic nucleotide. This is a very small molecule that rotates fast (low FP) . PDE8A1 catalyzes the hydrolysis of the phosphodiester bond in the cyclic nucleotide and frees the phosphate group. In a second step the free phosphate group is recognized by a specific phosphate-binding nanobead (Binding Agent) leading to the formation of a large complex, with restricted movement (high FP) . FP is proportional to PDE8A1 activity.Need us to run inhibitor screens or profile your compounds against PDE8A? Check out ourPhosphodiesterase Screening Services.This assay requires a fluorescent microplate reader capable of measuring fluorescence polarization (FP) and equipped with the required parts to read the FP signal. For more information FP technology, visit our Tech Note:FP, assay principles and applications.Note:As of December 2024, this protocol has been re-optimized for performance. Previous versions of this kit are available upon request.

• UniProt:

  O60658

• Applications:

  Useful for studying enzyme kinetics and screening small molecular inhibitors for drug discovery and HTS applications.

• Format:

  Catalog #NameAmountStorage60080PDE8A1, GST-tag*˃1 µg-80°C60200FAM-Cyclic-3′, 5′-AMP**1.2 nmoles**-80°C60393PDE Assay Buffer (Incomplete) 25 ml-20°C60390PDE Binding Agent100 µl4°C60391PDE Binding Agent Diluent (cAMP) 10 ml4°C827350.5 M DTT200 µl-20°C79685Low binding, black 96-well plate1Room Temp.* The concentration of protein is lot-specific and will be indicated on the tube containing the protein.**FAM-Cyclic-3′, 5′-AMP is provided as a powder. The vial will need to be resuspended in 600 µl of Complete PDE Assay Buffer before use.

• Shipping Conditions:

  -80°C

• Storage Conditions:

  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.

• Notes:

  MATERIALS OR INSTRUMENTS REQUIRED BUT NOT SUPPLIED:Fluorescent microplate reader capable to measure fluorescence polarization

• Contraindications:

  The final concentration of DMSO in the assay should not exceed 1%.Fluorescent compounds that have λex=470 nm and detection at λem=528 nm can interfere with the readouts.It is recommended that the compound alone is tested to determine any potential interference of the compound on the assay results.