PDE10A1 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. PDE10A is a dual substrate PDE highly expressed in striatal medium spiny neurons. PDE10A inhibitors can improve the cognitive symptoms of schizophrenia and exhibit potential therapeutic effect value for Huntington’s disease. PDE10A1 is located in the cytosol, whereas PDE10A2 is a membrane-associated protein. Its various functions make it a highly relevant target for the treatment of many disorders, and the use of PDE10A1 inhibitors will continue to open new therapeutic avenues.

• Description :

  The PDE10A1 Assay Kit is a fluorescence polarization (FP), homogeneous, 96-well assay kit designed for the screening and profiling of PDE10A1 (Phosphodiesterase 10A1) inhibitors. This assay takes advantage of a specific fluorescent phosphate-binding nanoparticle. The kit contains enough purified recombinant PDE10A1, fluorescent probe, PDE assay buffer, Binding Agent, and diluent for 400 reactions.Figure 1: Illustration of the PDE10A1 Assay Kit principle.The assay uses a fluorescein-labeled cyclic guanosine monophosphate (cAMP-FAM for PDE10A1), in which the phosphate group is engaged within the cyclic nucleotide. This is a very small molecule that rotates fast (low FP) . PDE10A1 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 PDE10A1 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.

• UniProt :

  Q9Y233

• Applications :

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

• Format :

  Catalog #NameAmountStorage60099PDE10A1, 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. Each 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 storedas directed.