Molecular Devices SpectraMax Paradigm User Manual page 120

SpectraMax Paradigm Multi-Mode Detection Platform User Guide
The GeneBLAzer technology provides a sensitive method to quantitate gene expression in
mammalian cells. GeneBLAzer Detection Kits enable in vivo (living cells) or in vitro (cell
lysates) detection of β-lactamase reporter activity using a special fluorescent substrate. The
GeneBLAzer assay system consists of two major components:
The β-lactamase reporter gene, bla(M), a modified form of the E. coli bla gene. When
fused to promoter sequences or to a gene of interest (in the context of a vector), the bla
(M) gene can be used as a reporter of promoter activity or gene expression in
mammalian cells, respectively.
CCF2, a FRET-based substrate permitting fluorescent detection of β-lactamase reporter
activity. CCF2 consists of a core (enzyme substrate site) linked to two fluorophores, 7-
hydroxycoumarin and fluorescein. If there is no bla expression, then the substrate
molecule remains intact. In this state, excitation of the coumarin results in fluorescence
resonance energy transfer to the fluorescein moiety and emission of green light.
However, in the presence of bla expression, the substrate is cleaved, separating the
fluorophores, and disrupting energy transfer. Excitation of the coumarin in the presence
of β-lactamase activity results in a blue fluorescence signal. The resulting blue:green ratio
provides a normalized reporter response.
For GeneBLAzer data, refer to the Invitrogen GeneBLAzer Detection Kits Instruction Manual,
25-0661. Background fluorescence should be determined using sample wells containing
medium plus serum without cells.
The ratio of blue and green fluorescence signal is calculated by dividing the 465 nm emission
(blue channel) reading by the 535 nm emission (green channel) reading:
Ra o =
The ratio obtained from the experimental sample is compared to the ratio obtained from the
applicable negative controls.
For more information about fluorescence intensity, see
on page
120
(signal at 465 nm - background at 465 nm)
(signal at 535 nm - background at 535 nm)
28.
Fluorescence Intensity Read Mode
5014038 E