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Luminometer
Protocol for Bright-Glo™
1.
INTRODUCTION
The
Reporter™Microplate Luminometer, combined with
Promega’s Bright-Glo™ Luciferase Assay System,
provides an easy to use, sensitive method in the quantitation
of gene expression. Transcriptional regulation, coupled
to the expression of a reporter gene, is regularly
used to study a wide range of physiological events.
A popular example is the analysis of receptor function
by quantifying the action of specific receptor response
elements on gene expression. Luciferase is a recommended
choice as a reporter because functional enzyme is
created immediately upon translation, and the assay
is swift, dependable and easy to perform1, 2.
Additionally, analysis using luciferase as the genetic
reporter is appropriate for laboratory automation
and high-throughput applications. For these reasons,
luciferase is widely used in the pharmaceutical and
biotechnology industries.
The
Bright-Glo™ Luciferase Assay System from Promega
Corporation has been developed specifically to maximize
the sensitivity of the assay reagent while providing
a luminescent signal half-life generally exceeding
25 minutes.
Broad
applicability of luciferase reagents is necessary
for wide use in homogenous, single-step assays and
in nonhomogeneous, two-step assays. The Bright-Glo™
reagent is compatible with most commonly used culture
media for mammalian cells (RPMI 1640, MEM?, DMEM and
F12, with or without added serum), and can tolerate
phenol red and organic solvents. Furthermore, the
reagent is tolerant of incomplete mixing, making it
suitable for multiwell plates.
2.
MATERIALS REQUIRED
- The
Reporter™ Microplate Luminometer (Turner BioSystems
P/N 9600-001)
- Bright-Glo™
Luciferase Assay System (Promega Catalog #'s E2610,
E2620, E2650)
- 8
channel 200m l Autopipettor
- 200m
l Pipette Tips
- Solid
White Assay Plate, 96-well, high binding, flat (VWR
P/N 29444-020)
3.
REAGENT PREPARATION
3.1
Bright-Glo™ Substrate: Use as supplied. Store
at -20° C, where it is stable for up to 6 months.
The substrate may also be stored at 4° C for up to
two weeks.
3.2
Bright-Glo™ Buffer: Use as supplied. Store below
25° C.
3.3
Bright-Glo™ Reagent: Transfer the contents of
one bottle of Bright-Glo™ Buffer to one bottle
of Bright-Glo™ Substrate. Mix by inversion until
the substrate is thoroughly dissolved. Use reconstituted
reagent on the same day of its preparation or store
at –70° C for up to one month.
Note:
The temperature of the Bright-Gloä Reagent should
be held constant at room temperature while quantitating
luminescence since luciferase activity is temperature
dependent. Reagent stored frozen after reconstitution
must be thawed below 25° C to ensure reagent performance.
Mix well after thawing. The simplest method for thawing
is placing the reagent in a water bath at room temperature.
4.0
Instrument Set-up
4.1
Turn on both the Reporter™ Microplate Luminometer
and the computer. Allow the instrument to warm up
for at least 10 minutes.
4.2
Double click on the Reporter icon to start the software.
4.3
In the upper left corner, click on ‘File’
then ‘New’.
4.4
Enter your information into the ‘Test’,
‘Operator’, ‘Plate No.’, and ‘Notes’
fields, as desired.
4.5
Click on the ‘Options’ button to open the
options screen
4.6
Select ‘Single Run’ or ‘Multiple Run’
as desired.
4.7
Click on each well that you want to be read. Wells
selected will be green. Unselected wells will be white.
To reduce the reading time, select only the wells
that will have the Bright-Glo reagent.
4.8
Click ‘OK’ when selections are completed.
5.0
Sample Analysis
5.1
Remove 96 well plate containing cell cultures from
the incubator. Note: For maximum reproducibility,
equilibrate cell cultures to room temperature before
adding reagent.
5.2
Add a volume of the Bright-Gloä reagent equal to that
of the culture medium in each well. For 96 well plates,
typically 100 ul of reagent is added to cells grown
in 100 ul of medium.
5.3
Press sample compartment door button on the front
of the Reporter™ Microplate Luminometer to open
the sample compartment.
5.4
Carefully insert 96 well plate with samples and reagent
into the tray. Avoid splashing or spilling samples.
5.5
Press button to close door.
5.6
Wait at least 2 minutes to allow for complete cell
lysis. However, for maximal light intensity, samples
should be analyzed within 15 minutes of reagent addition.
5.7
Click on ‘Start’ to begin sample readings.
You will be prompted to enter a file name for the
data file. Enter a file name and click on ‘Save’.
After you click on ‘Save’, the run will
begin.
5.8
Remove 96 well plate from the instrument when measurements
are complete and data are displayed on the main screen.
6.
REFERENCES
1
Ow, D.W. et al. (1986) Transient and stable
expression of the firefly luciferase gene in plant
cells and transgenic plants. Science 234, 856.
2
De wet, J.R. et al. (1987) Firefly luciferase gene:
structure and expression in mammalian cells. Mol.
Cell. Biol. 7, 725
CAUTION:
The
lyophilized Bright-Glo® Substrate contains dithiothreitol
(DTT) and is therefore classified as hazardous. The
reconstituted reagent is not known to present any
hazards as the concentration of DTT is less than 1%.
However, we recommend the use of gloves, lab coats
and eye protection when working with these or any
chemical reagents. Promega and Turner BioSystems assume
no liability for damage resulting from handling or
contact with these products.
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