E-Book Overview
A state-of-the-art collection of innovative methods for the use of luciferase and green fluorescent protein in analytical biochemistry, molecular toxicology, environmental monitoring, and cell-based assays. The contributors-all hands-on researchers who have perfected their protocols to a high degree of reproducibility-illustrate these powerful methods with a wide variety of applications ranging from the analytical determination (titer) of specific molecules to the cellular detection of a wide range of pollutants, to microbiological detection and analysis, and the molecular biological manipulation of lux, luc, and gfp structural genes. Step-by-step methods are also included for the construction of other recombinant organisms.
E-Book Content
1 Improvements in the Application of Firefly Luciferase Assays Sharon R. Ford and Franklin R. Leach 1. Introduction 1.1. Firefly Luciferase Assay Differs from Usual Enzyme Assays The firefly luciferase-based assay differs from most familiar enzyme-based determinations. Most enzyme assaysare 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 Striphosphate (ATP) concentrations, firefly luciferase is a stoichiometric reactant rather than a catalyst. In the case of the firefly luciferase reaction, AMP, PPi, CO*, 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. The two-step firefly luciferase reaction sequence is shown below. Step one forms an enzyme-bound luciferyl adenylate. Either MgATP or LH, (luciferin) can add first to the enzyme LUC. LH2 + MgATP + LUC c----) LUC-LH