Luciferase Reporter Assay

Luciferase is a generic term for the class of oxidative enzymes used in bioluminescence and is distinct from a photoprotein. Luciferase has been used as a sensitive reporter for studying gene regulation and function. Luciferase provides a 1000-fold increase in sensitivity in comparison to the standard chloramphenicol acetyltransferase (CAT) assay. Luciferase catalyzes the oxidative carboxylation of luciferin, a reaction with the highest efficiency of any known bioluminescence reaction.

The chemical reaction catalyzed by firefly luciferase takes place in two steps:

  1. luciferin + ATP → luciferyl adenylate + PPi
  2. luciferyl adenylate + O2 → oxyluciferin + AMP + light

Luciferase can be produced in the lab through genetic engineering for a number of purposes. Luciferase genes can be synthesized and inserted into organisms or transfected into cells. Mice, silkworms, and potatoes are just a few of the organisms that have already been engineered to produce the protein.

In the luciferase reaction, light is emitted when luciferase acts on the appropriate luciferin substrate. Photon emission can be detected by light sensitive apparatus such as a luminometer or modified optical microscopes. This allows observation of biological processes. Since light excitation is not needed for luciferase bioluminescence, there is minimal autofluorescence and therefore virtually background-free fluorescence. Therefore, as little as 0.02pg can still be accurately measured using a standard scintillation counter.

Luciferase Reporter Assay

Luciferase Reporter Assay at Creative BioMart can help you detect luciferase activity both in vitro. Luciferase is needed to extract from materials. Then CoA, ATP, Mg2+ and buffer are added to the lysate (Inclusion of CoA yields a nearly constant light emission rather than typical flash kinetics). The luminescent reaction is then triggered by an injection of luciferin, and the emitted light is recorded by a luminometer.

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