PDE4D2 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. PDE4 is the second most abundant PDE in cardiovascular tissues. PDE4, is a cAMP-specific PDE and the largest PDE subfamily with over 35 different isoforms and as such, the most widely characterized PDE isoenzyme. It is the predominant isoenzyme in the majority of inflammatory cells, and the airways smooth muscle and is implicated in inflammatory airways disease. PDE4 inhibitor roflumilast has been approved for the treatment of chronic obstructive lung disease.

• Description:

  The PDE4D2 Assay Kit is a fluorescence polarization (FP), homogeneous, 96-well assay kit designed for the screening and profiling of PDE4D2 (Phosphodiesterase 4D2) inhibitors. This assay takes advantage of a specific fluorescent phosphate-binding nanoparticle. The kit contains enough purified recombinant PDE4D2, fluorescent probe, PDE assay buffer, Binding Agent, and diluent for 100 reactions.Figure 1: Illustration of the PDE4D2 Assay Kit principle.The assay uses a fluorescein-labeled cyclic adenosine monophosphate (cAMP-FAM for PDE4D2), in which the phosphate group is engaged within the cyclic nucleotide. This is a very small molecule that rotates fast (low FP) . PDE4D2 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 PDE4D2 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 July 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 PDE4D2? Check out ourPhosphodiesterase Screening Services.This product has beencited 8 times.

• UniProt:

  Q08499-5

• Applications:

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

• Format:

  Catalog #NameAmountStorage60048PDE4D2*˃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°C0.5 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. 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 to 6 months from date of receipt when the materials are stored as directed.

• Notes:

  Troubleshooting GuideVisitbpsbioscience.com/assay-kits-faqfor detailed troubleshooting instructions. For further questions, email[email protected]

• 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.