Phosphodiesterases (PDEs) play an important role in dynamic regulation of cAMP and cGMP signaling. PDE4D is a regulator of airway smooth-muscle contractility, and has been identified as a potential risk predictor for ischemic stroke. Additionally, PDE4D has been associated with asthma pathophysiology and bone formation. The PDE4D gene encodes at least 9 different isoforms.
Intended Use:The TR-FRET PDE4D3 Assay Kit is designed for identification of inhibitors of PDE4D3 using TR-FRET (Time Resolved Fluorescence Resonance Energy Transfer) technology. The assay is based on the binding of phosphate groups from FAM-labeled nucleotide monophosphates generated by PDE4D3 and from terbium donors to the nanoparticle binding agent. The binding will result in energy transfer from terbium to FAM and the fluorescent intensity change can be measured using fluorescence reader.
Test Principle:The TR-FRET PDE4D3 Assay Kit comes in a convenient 96-well format, with purified PDE4D3 enzyme, fluorescently labeled PDE4D3 substrate (cAMP), binding agent, and PDE assay buffer for 100 enzyme reactions. Using this kit, only two simple steps on a microtiter plate are required for the PDE4D3 activity assay. First, the fluorescent-labeled cAMP is incubated with a sample containing PDE4D3 for 1 hour. Second, a binding agent and a terbium donor are added to the reaction mix and incubated for 1 hour. Then, fluorescence intensity can be measured using a fluorescence reader.
Materials or Instruments Required But Not Supplied:Fluorescent microplate reader capable of measuring Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET)
Applications:Suitable for screening small molecular inhibitors for drug discovery and HTS applications.
Storage and Stability:Store other components at 4°C. Stable for 6 months. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
