The Chemistry of Bioluminescent Reporter Assays
Firefly luciferase is the most widely used bioluminescent reporter because its enzyme activity is closely coupled to protein synthesis, and the luminescence assay is rapid, convenient and sensitive. Although various assay formulations for firefly luciferase have been described, the most widely used contains coenzyme A in addition to beetle luciferin and ATP. In a 1-10 second measurement, this assay provides linearity over a 100 million-fold concentration range with sensitivity greater than 10-20 moles of enzyme. Recently, Renilla luciferase has also become widely used as a genetic reporter, although primarily as a co-reporter to firefly luciferase. Assay of Renilla luciferase is also rapid and linear, but the sensitivity is limited somewhat by autoluminescence. An assay format called the Dual-Luciferase™ Reporter (DLR™) Assay has been designed to sequentially quantitate both firefly and Renilla luciferases from a single sample. The integration of the two luciferase assays provides an efficient means for incorporating an internal control into reporter measurements, or for analyzing two separate events in the same system. Bacterial luciferase, although the first luciferase to be used as a reporter, is generally used to provide autonomous luminescence in bacterial systems through expression of the lux operon. Ordinarily it is not useful for analysis in eukaryotic systems.
Promega Notes 65, 12–12.
Keith V. Wood
Publication Date: 1998