Cell Biolabs’ Pyrophosphate Assay Kit measures total pyrophosphate within biological samples. Pyrophosphate (and phosphoenolpyruvate) is converted by PPDK to pyruvate + adenosine triphosphate (ATP) and phosphate. Pyruvate is converted by pyruvate oxidase in the presence of phosphate and oxygen into acetyl phosphate, carbon dioxide, and hydrogen peroxide. The resulting hydrogen peroxide is then detected with a highly specific fluorometric probe. Horseradish peroxidase catalyzes the reaction between the probe and hydrogen peroxide, which bind in a 1:1 ratio. Samples are compared to a known concentration of pyrophosphate standard within the 96-well microtiter plate format. Samples and standards are incubated for 30 minutes and then read with a standard 96-well fluorometric plate reader.