PDE4C 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 most 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 (COPD) .

• Description:

  The PDE4C1 Assay Kit is a fluorescence polarization (FP), homogeneous, 96-well assay kit designed for the screening and profiling of PDE4C1 (Phosphodiesterase 4C1) inhibitors. This assay takes advantage of a specific fluorescent phosphate-binding nanoparticle. The kit contains enough purified recombinant PDE4C1, fluorescent probe, PDE assay buffer, Binding Agent, and diluent for 100 or 400 reactions.Figure 1: Illustration of the PDE4C1 Assay Kit principle.The assay uses a fluorescein-labeled cyclic guanosine monophosphate (cAMP-FAM for PDE4C1), in which the phosphate group is engaged within the cyclic nucleotide. This is a very small molecule that rotates fast (low FP) . PDE4C1 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 PDE4C1 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 January, 2025, this protocol has been re-optimized for performance. Previous versions of this kit are available upon request

• UniProt:

  Q08493

• Applications:

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

• Format:

  96 reactionsCatalog #ComponentAmountStorage60044PDE4C1, 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 Temp384 reactionsCatalog #ComponentAmountStorage60044PDE4C1, GST-tag*˃1 µg-80°C60200FAM-Cyclic-3′, 5′-AMP**2 x 1.2 nmoles**-80°C60393PDE Assay Buffer (Incomplete) 25 ml-20°C60390PDE Binding Agent250 µl4°C60391Binding Agent Diluent (cAMP) 25 ml4°C827350.5 M DTT200 µl-20°C79961Low binding, black 384-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 1200 µl of Complete PDE Assay Buffer before use.

• Shipping Conditions:

  -80°C (Dry Ice)

• 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)