Bio-Rad Fluorescent Assay Kits - Manual (Page 3)

Section 1
Introduction
ß-galactosidase from E. coli, encoded by the lac Z gene, is one of the
most versatile reporters for gene expression studies. In addition to its use
as a reporter, ß-galactosidase is used to normalize cell variability of other
reporter assays, such as the chloramphenicol acetyltransferase (CAT) and
firefly luciferase assay.
1 We have developed a quantitative fluorescent
assay to determine the level of expression using 4-methylumbelliferyl-
galactopyranoside (MUG) as substrate. ß-galactosidase hydrolyzes the
fluorogenic substrate resulting in release of the fluorescent molecule
4-methylumbelliferone (4MU). Fluorescence of the 4-methylumbellifer-
one is then measured on a fluorometer using an excitation wavelength of
360 nm and emission wavelength of 460 nm.
The assay is initiated by adding 2–4 µg of total protein to 500 µl of
assay buffer. Samples are incubated in a 37 °C water bath for 15 minutes,
during which time the ß-galactosidase hydrolyzes the colorless substrate
to a highly fluorescent compound. The reaction is terminated by addition
of 1x Stop Buffer. The amount of product formation is measured in a flu-
orometer.
Section 2
Kit Components
2.1 Contents
The FluorAce ß-galactosidase Reporter Assay Kit contains reagents
for 200 x 1.5 ml or 2000 x 150 µl reactions, 4MU calibration curve, and
purified ß-galactosidase as the positive control. The fluorometer must be
equipped with 360 nm excitation and 460 nm emission filters.
1
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