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The Chemistry of Bioluminescent
Reporter Assays
By Keith V. Wood
Promega Corporation
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-LuciferaseTM Reporter
(DLRTM) 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.
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