The PDE5A1 Assay is a fluorescence polarization (FP), homogeneous 384-well assay designed for the screening & profiling of PDE5A1 inhibitors. This assay takes advantage of a specific fluorescent phosphate-binding nanoparticle. The kit contains enough purified recombinant PDE5A1, fluorescent probe, PDE assay buffer, Binding Agent, and diluent for 400 reactions. This assay requires a fluorescent microplate reader capable of measuring fluorescence polarization (FP) to read the FP signal. For more information on the principles of FP, visit fp_assays.pdf ().

Figure 1: Illustration of the PDE5A1 assay principle.
The assay uses a fluorescein-labeled cyclic guanine monophosphate (cGMP-FAM), in which the phosphate group is engaged within the cyclic nucleotide. This is a very small molecular that can rotate fast (low FP). PDE5A1 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 large complex, with restricted movement (high FP). FP is proportional to PDE activity.

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