Phosphodiesterases (PDEs) play an important role in dynamic regulation of cAMP and cGMP signaling. PDE7B is highly expressed in the pancreas and in other various tissues. Inhibition of PDE7B leads to induction of apoptosis.
Phosphodiesterases catalyze the hydrolysis of the phosphodiester bond in dye-labeled cyclic monophosphates. Beads selectively bind the phosphate group in the nucleotide product. This increases the size of the nucleotide relative to unreacted cyclic monophosphate. In the polarization assay, dye molecules with absorption transition vectors parallel to the linearly-polarized excitation light are selectively excited. Dyes attached to the rapidly-rotating cyclic monophosphates will obtain random orientations and emit light with low polarization. Dyes attached to the slowly-rotating nucleotide-bead complexes will not have time to reorient and therefore will emit highly polarized light.
The PDE7B Assay Kit is designed for identification of PDE7B inhibitors using fluorescence polarization. The assay is based on the binding of a fluorescent nucleotide monophosphate generated by PDE7B to the binding agent.
Test Principle:The PDE7B inhibitor screening assay kit comes in a convenient 96-well format, including purified PDE7B enzyme, fluorescently labeled PDE7B substrate (cAMP), binding agent, and PDE assay buffer for 100 enzyme reactions. The key to the PDE7B Assay Kit is the specific binding agent. Using this kit, only two simple steps on a microtiter plate are required for PDE7B reactions. First, the fluorescently labeled cAMP is incubated with a sample containing PDE7B for 1 hour. Second, a binding agent is added to the reaction mix to produce a change in fluorescent polarization that can then be measured using a fluorescence reader.
Storage and Stability:Store *137350D at -20ºC. Store *137350B and *137350C at -70ºC. Store all other components at 4°C. Stable for 6 months after receipt. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
