PDE5A1 Assay Kit
The Phosphodiesterase 5A1 (PDE5A1) Assay Kit is a fluorescence polarization (FP), homogeneous 384-well assay kit 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.
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.
This assay requires a fluorescent microplate reader capable of measuring fluorescence polarization (FP) to read the FP signal. For more information FP technology, visit our Tech Note: FP, assay principles and applications.
This product has been cited 9 times.
Fluorescent plate reader capable of measuring fluorescence polarization.
Catalog # | Component | Amount | Storage |
60050 | PDE5A1, GST-Tag* | >1 µg | -80°C |
60201 | 20 µM FAM-Cyclic-3′, 5′-GMP | 50 µl | -80°C |
60393 | PDE Assay Buffer | 25 ml | -20°C |
60390 | Binding Agent | 250 µl | +4°C |
60392 | cGMP Binding Agent Diluent | 25 ml | +4°C |
79961 | Low binding, black 384-well plate | 1 | Room temp. |
* The concentration of protein is lot-specific and will be indicated on the tube containing the protein.
Phosphodiesterases (PDEs) play an important role in the dynamic regulation of the second messengers cAMP and cGMP signaling, by hydrolyzing them. PDE5A1, also known as cGMP-inhibited phosphodiesterase, is found in cerebellum, kidney, pancreas, lung, and vascular smooth muscle, and it has been implicated in cardiovascular function and fertility. Several inhibitors have been identified, such as sildenafil, vardenafil and tadalafil, for the treatment of pulmonary hypertension and erectile dysfunction. The reported side effects on vision may reveal new roles for PDE5 in the retina, so further studies into the functions of PDE5A1 and development of new inhibitors will be advantageous.
Maurice DH., Front. Biosci. 2005; 10: 1221-1228