Improvements in the Application of Firefly Luciferase Assays

The firefly luciferase-based assay differs from most familiar enzyme-based determinations. Most enzyme assays are based either on the production of a product or the disappearance of a substrate. Usually the compound measured is stable so that its concentration can be determined after a specific time. At low adenosine 5′triphosphate (ATP) concentrations, firefly luciferase is a stoichiometric reactant rather than a catalyst. In the case of the firefly luciferase reaction, AMP, PPi, CO2, and oxyluciferin are typical products that accumulate, but the product that is most often and most easily determined is light. The photons of light are not accumulated in the measuring technique unless film or some electronic summation procedure is used in photon counting.