PDE3B 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. PDE3 is a major cAMP-hydrolyzing PDE in heart muscle, vascular smooth muscle and oocytes. The PDE3 family in mammals consists of two members, PDE3A and PDE3B. The PDE3 isoforms are structurally similar, containing an N-terminal domain important for their localization. PDE3 expression has been described as a biomarker for sensitivity to the PDE3-inhibitor Zardaverine in different types of cancer. PDE3 inhibitors have been developed as pharmaceuticals, but their use is limited due to the increase in arrhythmia, and they can increase mortality in some applications. However, the PDE3 inhibitors enoximone and milrinone can be used as a rescue drug in life-threatening bronchial asthma, indicating the therapeutical potential of targeting PDE3.

• Description :

  The PDE3B Assay Kit is a fluorescence polarization (FP), homogeneous, 96-well assay kit designed for the screening and profiling of PDE3B (Phosphodiesterase 3B) inhibitors. This assay takes advantage of a specific fluorescent phosphate-binding nanoparticle. The kit contains enough purified recombinant PDE3B (amino acids 592-1112 (end) ), fluorescent probe, PDE assay buffer, Binding Agent, and diluent for 100 reactions.Figure 1: Illustration of the PDE3B Assay Kit principle.The assay uses a fluorescein-labeled cyclic adenosine monophosphate (cAMP-FAM for PDE3B), in which the phosphate group is engaged within the cyclic nucleotide. This is a very small molecule that rotates fast (low FP) . PDE3B 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 PDE3B activity.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 November 2024, this protocol has been re-optimized for performance. Previous versions of this kit are available upon request.Need us to run inhibitor screens or profile your compounds against PDE3B? Check out ourPhosphodiesterase Screening Services.

• UniProt :

  Q14432

• Applications :

  Study enzyme kinetics and screen small molecule inhibitors for drug discovery in high throughput screening (HTS) applications.

• Format :

  Catalog #NameAmountStorage60031PDE3B, 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°C60391Binding 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 :

  This assay kit will perform optimally for up to6 monthsfrom date of receipt when the materials are stored as directed.

• Notes :

  Materials Required but Not SuppliedAdjustable micropipettor and sterile tipsRotating or rocker platformFluorescent microplate reader capable of measuring fluorescence polarization (λex=470 (5 nm bandwidth) and detection at λem=528 (10 nm bandwidth)

• 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.