PDE4B (Rat) Assay Kit

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 abundantPDEin cardiovascular tissues. PDE4, is a cAMP-specific PDE and the largest PDE subfamily with over 35 differentisoformsand as such, the most widely characterized PDEisoenzyme. Inhibitors of PDE4 are being used for the treatment of psoriasis, COPD (chronic obstructive pulmonary disease) and atopic dermatitis. PDE4B is the most abundant PDE4 in the brain and it is involved in cognitive function and memory, and diseases such as schizophrenia and neuroinflammation. Evidence points for the formation of a PDE4B/DISC1 (Disrupted in Schizophrenia 1) complex that regulates PKA (Protein kinase A) phosphorylation of NDEL1 (nuclear distribution factor-E-like) and its interaction with LIS1 (lissencephaly 1), and thus impacts synaptic function. In the context of neuroinflammation PDE4B is linked to the NF-κB (necrosis factor kappa B) signaling pathway via the regulation of cAMP levels in cells. High levels of PDE4B have also been found in colon cancer and hematological disorders such as CLL (chronic lymphocytic leukemia) . The development of PDED4B specific inhibitors and a deeper understanding of the role of each PDE4B isoform in health and disease will prove beneficial for treatment of PDE4B related disorders.

The PDE4B (Rat) Assay Kit is a fluorescence polarization (FP), homogeneous, 96-well assay kit designed for the screening and profiling of PDE4B (Phosphodiesterase 4B) inhibitors. This assay takes advantage of a specific fluorescent phosphate-binding nanoparticle. The kit contains enough purified recombinant rat PDE4B, fluorescent probe, PDE assay buffer, Binding Agent, and diluent for 100 reactions.Figure 1: Illustration of the PDE4B (Rat) Assay Kit principle.The assay uses a fluorescein-labeled cyclic adenosine monophosphate (cAMP-FAM for PDE4B), in which the phosphate group is engaged within the cyclic nucleotide. This is a very small molecule that rotates fast (low FP) . PDE4B 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 PDE4B 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 December 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 PDE4B (Rat) ? Check out ourPhosphodiesterase Screening Services.

P14646

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

Catalog #NameAmountStorage60049Rat PDE4B, 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. Vial will need to be resuspended in 600 µl of Complete PDE Assay Buffer before use.

-80°C

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

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