PDE3A TR-FRET Assay Kit

• Background:

  Phosphodiesterases (PDEs) play an important role in dynamic regulation of cAMP and cGMP signaling. PDE3A, also known as cGMP-inhibited phosphodiesterase, is activated by protein kinase A and B, and can be inhibited by cGMP. It has been implicated in cardiovascular functions by regulating the contractility of the heart muscle and vascular smooth muscle, and platelet aggregation. It has also been linked to fertility based on its expression in oocytes. The use of inhibitors for PDE3A and PDE3B showed some promise for cardiovascular disease treatment but can result in side effects such as arrhythmia. PDE3A has been found in lung and breast cancer, where it mediates cancer cell stemness by downregulating the cAMP/PKA pathway, and its level links to clinical prognosis. The use of PDE3A inhibitors has been shown to result in reduction of breast cancer metastasis in animal models and may prove a powerful tool in cancer therapy.

• Description:

  The PDE3A TR-FRET Assay Kit is designed to provide fast and easy identification of inhibitors of PDE3A (phosphodiesterase 3A) using Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) . The PDE3A TR-FRET Assay Kit comes in a convenient well format, with enough recombinant purified PDE3A enzyme, fluorescently labeled PDE substrate (cAMP), binding agent, and PDE assay buffer for 96 enzyme reactions.Figure 1: Illustration of the assay principle.The reaction uses a fluorescein-conjugated (FAM) cyclic monophosphate nucleotide.  Phosphodiesterase PDE3A catalyzes the hydrolysis of the phosphodiester bond in the cyclic monophosphate nucleotide, releasing the phosphate group for binding. The phosphate group binds to a “Binding Agent” (BA) that is recognized by terbium-labeled donor beads. This results in energy transfer from the terbium to FAM, which emits a fluorescent signal at 520 nm. If unbound to the phosphate group, the terbium-labeled beads emit at λ=490 nm. The fluorescent intensity is measured using a fluorescence plate reader capable of TR-FRET reading and an increase in 520 nm corresponds directly to the activity of PDE3A. In this assay, the intensity of the signal at 520 nm is directly proportional to PDE3A enzymatic activity.Need us to run inhibitor screens or profile your compounds against PDE3A? Check out ourPhosphodiesterase Screening Services.This product has beencited 1 time.

• UniProt:

  Q14432

• Applications:

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

• Format:

  94-WellCatalog #NameAmountStorage60032PDE3A, (669-end), His-GST-tags*>1 µg-80°C60200FAM-Cyclic-3’ , 5’AMP**1.2 nmoles**-80°C60393PDE Assay Buffer (Incomplete) 25 ml-20°C60394Tb Donor50 µl-80°C82782PDE Binding Agent (TR-FRET) 200 µl+4°C78422Binding Buffer A20 ml+4°C78423Binding Buffer B20 ml+4°C827350.5 M DTT200 µl-20°C79685Low binding, black 96-well plate1Room Temp384-WellCatalog #NameAmountStorage60032PDE3A, (669-end), His-GST-tags*>1 µg-80°C60200FAM-Cyclic-3’ , 5’AMP**2 x 1.2 nmoles**-80°C60393PDE Assay Buffer (Incomplete) 25 ml-20°C60394Tb Donor50 µl-80°C82782PDE Binding Agent (TR-FRET) 200 µl+4°C78422Binding Buffer A20 ml+4°C78423Binding Buffer B20 ml+4°C827350.5 M DTT200 µl-20°C79961Low binding, black 384-well plate1Room Temp*The concentration of the protein is lot-specific and will be indicated on the tube.**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 to6 monthsfrom date of receipt when the materials are stored as directed.

• Notes:

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

• Contraindications:

  The final concentration of DMSO in the assay should not exceed 1%.Compounds that are fluorescent may interfere with the results, depending on their spectral excitation and emission properties.It is recommended that the compound alone is tested to determine any potential interference of the compound on the assay results.