Veritas™
Microplate Luminometer Method for
Promega Beta-Glo®
1. INTRODUCTION
The Turner BioSystems Veritas™ Microplate
Luminometer in combination with Promega's Beta-Glo® Assay
System provides a reliable, homogeneous method to quantitate ß-galactosidase
expression in mammalian cells. Promega's Beta-Glo® Assay
System generates a bright, glow-type signal that remains stable for more
than 4 hours. The prolonged luminescence allows for batch processing of
multiple plates. The Beta-Glo® Assay System couples the
ß-galactosidase cleavage of 6-O-ß-galactopyranosyl-luciferin
with the firefly luciferase reaction to generate light1. The
amount of light detected by the Veritas™ Microplate Luminometer is
proportional to the amount of
ß-galactosidase present (Figure 1).
The extended dynamic range of the Veritas™
Microplate Luminometer allows the user to easily measure very dim and
very bright samples on the same plate using the Beta-Glo® Assay System. A pre-installed Beta-Glo® template on the
Veritas™ facilitates quick set-up. The Veritas™ Microplate Luminometer
detects as little as 30 fg ß-galactosidase using Beta-Glo® Reagent. Measurements are linear from 30 fg to 1 ng ß-galactosidase
or more than 4 orders of magnitude (Figure 1).
Figure 1. Beta-Glo® Assay performed on the Veritas™
Microplate Luminometer. ß-galactosidase was diluted in 25 mM HEPES
buffer containing 0.1% gelatin. Following the addition of Beta-Glo® Reagent, the microplate was incubated at room temperature for 60 minutes
before measurement.
The Beta-Glo™ Reagent is compatible
with commonly used culture media for mammalian cells (RPMI 1640, MEMa,
DMEM, and Ham's F12) containing 0—10% serum. The luminescent signal
is affected by the presence of phenol red, temperature changes and organic
solvents. Results should be compared only between samples measured with
similar media/serum mixtures. For optimal performance, minimize the presence
of phenol red and organic solvents (i.e. DMSO), which will otherwise decrease
luminescence.
2. MATERIALS REQUIRED
- Veritas™ Microplate Luminometer
(P/N 9100-000)
- 96-well plates, white (E&K Scientific
EK-25075)
- Beta-Glo® Assay System
(Promega Corporation E4720, E4740, or E4780)
- p200 pipette and pipette tips
3. EXPERIMENT PROTOCOL
3.1 Reagent Preparation
Beta-Glo® Assay Substrate:
Use as supplied. Store at -20°C.
Beta-Glo® Assay Buffer:
Use as supplied. Store at -20°C.
Beta-Glo® Assay Reagent:
Transfer the contents of one bottle of Beta-Glo® Assay
Buffer to one bottle of Beta-Glo® Assay Substrate. Mix by inversion
until the substrate is thoroughly dissolved. Use reconstituted reagent
on the same day it is prepared to generate best performance. Reconstituted
reagent may be stored at 22°C for up to 2 days with < 20% loss of potency and 4°C or -20°C for up to 7 days with < 10% loss of potency. Store reagent away from light.
Note: The temperature of the Beta-Glo® Assay Reagent should be held constant at room temperature while quantifying
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.
3.2 Instrument Setup
3.2.1 Double click on the Veritas™
icon to start the software.
3.2.2 Click on "Run Promega Protocol" from the "Welcome
to Veritas" dialog box.
3.2.3 Select "BetaGlo" from the list of Promega protocols.
3.2.4 Enter your information in the "Experiment", Operator",
"Plate No.", and "Notes" fields in the "Main
Dialog Box".
3.2.5 Click "Options" from the "Main Dialog Box"
to select the wells to be read and modify the number of runs. Once modified,
click on "Apply Changes" and return to the "Main Dialog
Box".
3.3 Sample Analysis
3.3.1 Remove the 96-well plate containing
cell cultures from the incubator.
Note: For maximum reproducibility, equilibrate cell cultures to
room temperature before adding reagent.
3.3.2 Add a volume of the Beta-Glo® Assay Reagent equal
to that of the culture medium in each well. For 96-well plates, typically
100 µL of reagent is added to cells grown in 100 µL of medium.
For optimal results, do not reduce the volume of reagent to less than
a 1:1 ratio with the volume of medium.
3.3.3 Use a plate shaker to mix the sample contents for 30 seconds.
Thorough mixing is necessary for maximum reproducibility.
3.3.4 Allow the sample to incubate at room temperature for at least
30 minutes.
Note: The initial ramp-up period for the luminescent signal to
reach maximum light intensity is 30—60 minutes. Between 30—60
minutes, the rate of increase in luminescence is < 20% per
10-minute period. The change in luminescent signal between 60—240
minutes is <10% per 60-minute period.
3.3.5 Insert the plate into the Veritas™ Microplate Luminometer
and click on "Start" to begin assay. RLU values measured by
the Veritas™ Microplate Luminometer will appear in the Excel spreadsheet
after all the selected wells in each row have been read. If you encounter
an error message, refer to the troubleshooting guide for more information.
Note: Opening another Excel spreadsheet while the Veritas™
reads your sample plate may cause loss of data and is not recommended.
3.3.6 Once the measurements are complete you can access Excel to analyze
your data.
Note: Please remove your plate after measurement.
4. REFERENCES
1. Geiger, R. et al. (1992) A new ultra
sensitive bioluminogenic enzyme substrate for ß-galactosidase.
Biol. Chem. Hoppe-Seyler 373, 1187—91.
5. ABOUT PROMEGA CORPORATION
Beta-Glo is a trademark of Promega Corporation and is registered with
the U.S. Patent and Trademark Office.
Orders for Promega's products may be placed by:
Phone: (800) 356-9526
Fax: (800) 356-1970
E-mail: custserv@promega.com
www.promega.com
Mailing Address:
Promega Corporation
2800 Woods Hollow Rd.
Madison, WI 53711
USA
6. ABOUT TURNER BIOSYSTEMS
Veritas is a trademark of Turner BioSystems, Inc.
Orders
for Turner BioSystems' products may be placed by:
Phone: (408)
636-2400
Toll Free: (888) 636-2401 or
Fax: (408) 737-7919
Contact us
via our contact
form
www.turnerbiosystems.com
Mailing Address:
Turner BioSystems, Inc.
645 N. Mary Avenue
Sunnyvale, CA 94085
CAUTION: The lyophilized Beta-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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