Revised 915
TM058
TECHNICAL MANUAL
DualGlo® Luciferase
Assay System
Instructions for use of Products
E2920 E2940 and E2980
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1 Description 2
2 Product Components and Storage Conditions 2
3 Performing the DualGlo® Luciferase Assay 5
3A General Considerations 5
3B Reagent Preparation 6
3C Assay Procedure 7
4 Related Products 8
5 References 11
6 Appendix 11
6A Overview of the DualGlo® Luciferase Assay System 11
6B Development of the Assay 12
Quenching Eff ect 14
6C Data Analysis 16
Background Subtraction 16
Normalizing Ratios 16
Relative Response Ratios 17
6D Conditions Aff ecting Assay Performance 18
Culture Medium 20
Serum 21
Phenol Red 22
Organic Solvents 23
Temperature 24
6E Summary of Changes 25
All technical literature is available at wwwpromegacomprotocols
Visit the web site to verify that you are using the most current version of this Technical Manual
Email Promega Technical Services if you have questions on use of this system techserv@promegacom
DualGlo® Luciferase Assay System
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1 Description
In cell biology research and pharmaceutical discovery it is common to test a wide variety of experimental conditions
or a large number of chemical compounds for their eff ects on cellular physiology (12) Traditionally the ease and
sensitivity of fi refl y luciferase assays have made it relatively simple to monitor the upregulation of genetic elements
However it has been more diffi cult to measure downregulation of genes because of the diffi culty in discriminating
between cell death and cellular downregulation Normalizing the expression of an experimental reporter to the
expression of a control reporter can help diff erentiate between specifi c and nonspecifi c cellular responses This
normalization can also control for transfection effi ciencies
Firefl y and Renilla luciferases are widely used as coreporters for these normalized studies because both assays are
quick easy and sensitive Firefl y luciferase is a 61kDa and Renilla luciferase a 36kDa protein (3–5) Both are
monomeric and neither requires posttranslational processing so they can function as genetic reporters immediately
upon translation
The DualGlo® Luciferase Assay System(a–c) is designed to allow highthroughput analysis of mammalian cells containing
genes for fi refl y and Renilla luciferases grown in 96 or 384well plates (Figure 1) The DualGlo® Luciferase Reagent can
be added directly to cells in growth medium without washing or preconditioning This reagent induces cell lysis and acts as
a substrate for fi refl y luciferase which has a halflife of approximately 2 hours Addition of the DualGlo® Stop & Glo®
Reagent quenches the luminescence from the fi refl y reaction by at least 10000fold and provides the substrate for Renilla
luciferase in a reaction that can also be read within 2 hours (with a similar retention in signal) The DualGlo® Luciferase
Assay System is designed to work in growth media commonly used for mammalian cells with or without added serum For
an overview and information on the development of the DualGlo® Luciferase Assay System see Sections 6A and B
To see articles that cite the use of the DualGlo® Luciferase Assay System visit
wwwpromegacomresourcestoolscitations
2 Product Components and Storage Conditions
PRODUCT SIZE CAT#
DualGlo® Luciferase Assay System 10ml E2920
Each system contains suffi cient components to prepare 10ml of each reagent Includes
• 10ml DualGlo® Luciferase Buff er
• 1 vial DualGlo® Luciferase Substrate (lyophilized)
• 10ml DualGlo® Stop & Glo® Buff er
• 100µl DualGlo® Stop & Glo® Substrate
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PRODUCT SIZE CAT#
DualGlo® Luciferase Assay System 100ml E2940
Each system contains suffi cient components to prepare 100ml of each reagent Includes
• 100ml DualGlo® Luciferase Buff er
• 1 vial DualGlo® Luciferase Substrate (lyophilized)
• 100ml DualGlo® Stop & Glo® Buff er
• 1000µl DualGlo® Stop & Glo® Substrate
PRODUCT SIZE CAT#
DualGlo® Luciferase Assay System 10 × 100ml E2980
Each system contains suffi cient components to prepare 1000ml of each reagent Includes
• 10 × 100ml DualGlo® Luciferase Buffer
• 10 vials DualGlo® Luciferase Substrate (lyophilized)
• 10 × 100ml DualGlo® Stop & Glo® Buffer
• 10 × 1000µl DualGlo® Stop & Glo® Substrate
Storage Conditions Store the DualGlo® Stop & Glo® Substrate and the lyophilized DualGlo® Luciferase Substrate
at –20°C The substrates also may be stored at 4°C for up to two weeks Store the DualGlo® Stop & Glo® Buff er and
the DualGlo® Luciferase Buff er below 25°C Buff er storage at room temperature is recommended to prevent the need
for temperature equilibration when the reagents are reconstituted Use the reconstituted DualGlo® Luciferase Reagent
on the day it is prepared or store at –70°C after preparation for up to six months Prepare the DualGlo® Stop & Glo®
Reagent on the day it is to be used
Caution The lyophilized DualGlo® Luciferase Substrate contains dithiothreitol (DTT) and is therefore classified
as hazardous The reconstituted reagent is not known to present a hazard 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 and all
chemical reagents In addition the DualGlo® Stop & Glo® Substrate contains a highly volatile solvent Please pipet
carefully and close the cap tightly after use
Note If DualGlo® Stop & Glo® Buff er precipitates upon freezing it can be resolubilized without aff ecting solution
performance To resolubilize precipitate store the DualGlo® Stop & Glo® Buff er at room temperature for 3 days or at
4°C for 2 weeks before use Precipitate can also be resolubilized by heating to 37°C for up to 2 hours with vigorous
shaking DualGlo® Stop & Glo® Buff er should be equilibrated to room temperature for use

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9
891MA
Add DualGlo® Luciferase
Assay Reagent to the plate
Incubate at 20–25°C for
10 minutes–2 hours
Incubate at 20–25°C for
10 minutes–2 hours
Measure firefly luminescence
Add DualGlo® Stop & Glo®
Reagent to the plate
Measure Renilla luminescence
Calculate ratio of fireflyRenilla
luminescence for each well
Normalize the sample well ratio
to the ratio from a control
(or series of control) well(s)
Figure 1 DualGlo® Luciferase Assay protocol Firefl y luciferase is produced measured and quenched followed by
Renilla luciferase in the same well
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3 Performing the DualGlo® Luciferase Assay
3A General Considerations
The DualGlo® Luciferase Assay System is designed for use in mammalian cell culture medium and is optimized for use
with the following types of media containing 0–10 serum RPMI 1640 DMEM MEM and F12 The reagents have
been developed so that the signals for fi refl y and Renilla luciferases are relatively stable and have a halflife of
approximately 2 hours Mediasera combinations aff ect the stability of each luminescent signal so experimental
determination of assay performance is recommended for mediasera combinations not listed above (see Section 6D)
The luminescence signal of each reporter also can be aff ected by the presence of phenol red organic solvents or changes
in temperature (Section 6D)
Because the luminescent signals are aff ected by assay conditions raw results should be compared only between samples
measured at the same time and using the same mediumserum combination Incorporation of consistent control wells
on each plate provides the ability to calculate a normalized fi refl y luminescenceRenilla luminescence ratio for each
sample well These normalized ratios will remain essentially constant (±10) for samples in a plate measured during
the recommended 2hour measurement window Incorporating positive and negative control wells within a plate or
experiment provides the ability to calculate a Relative Response Ratio (RRR) The RRR can be used to compare results
between experiments that do not use the same mediasera combination or have been aff ected by changes in
temperature or other variables (see Section 6C)
Because a small amount of time is required for complete cell lysis and enzyme equilibration the reagents of the
DualGlo® Luciferase Assay System should be added to plates 10 minutes before quantifying luminescence For
maximal light intensity samples should be measured within 2 hours of reagent addition The DualGlo® Luciferase
Reagents are not designed for use with the automated injectors that are integrated into some luminometers as
excessive reagent foaming may occur
To achieve linear assay performance at low light levels the background luminescence must be subtracted from all
readings Some luminometers also require verifi cation of linear response at high light levels (consult your luminometer
instrument manual for usage information)
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3B Reagent Preparation
1 Transfer the contents of one bottle of DualGlo® Luciferase Buff er to one bottle of DualGlo® Luciferase Substrate
to create the DualGlo® Luciferase Reagent Mix by inversion until the substrate is thoroughly dissolved
2 Calculate the amount of DualGlo® Stop & Glo® Reagent needed to perform the desired experiments Dilute the
DualGlo® Stop & Glo® Substrate 1100 into an appropriate volume of DualGlo® Stop & Glo® Buff er in a new
container
Example If 6ml of DualGlo® Stop & Glo® Reagent is needed dilute 60µl of DualGlo® Stop & Glo® Substrate into
6ml of DualGlo® Stop & Glo® Buff er For Cat# E2940 and E2980 if the entire bottle of DualGlo® Stop & Glo®
Buff er is to be used at one time transfer the entire contents of the DualGlo® Stop & Glo® Substrate into the buff er
For Cat# E2920 combine 10ml of DualGlo® Stop & Glo® Buff er and 100µl of DualGlo® Stop & Glo® Substrate
Notes
1 Assay reagents are stable at room temperature for several hours (see Notes 2 and 3) Freezing the reagent can reduce
the loss of activity of the DualGlo® Luciferase Reagent Do not thaw the reconstituted reagent at temperatures above
25°C Mix well after thawing The most convenient and eff ective method for thawing is to place the reagent in a room
temperature water bath Prepare only the amount of DualGlo® Stop & Glo® Reagent required For best results
prepare the DualGlo® Stop & Glo® Reagent immediately before use
2 DualGlo® Luciferase Reagent Stability Liquid reagent has approximately a 10 loss of fi refl y RLU after 8 hours
at room temperature and after 48 hours at 4°C Frozen reagent has approximately a 10 loss of fi refl y RLU after
1 week at –20°C and after 6 months at –70°C Do not store the reagent at –20°C for longer than 1 week The
reagent can be exposed to 5 freezethaw cycles with approximately a 15 loss in fi refl y RLU Holding or storing
the reconstituted reagent may cause Renilla RLU to rise
3 Approximate stability of DualGlo® Stop & Glo® Reagent after reconstitution 81 loss after 8 hours at room
temperature 85 loss after 24 hours at 4°C We recommend that the DualGlo® Stop & Glo® Reagent always be
prepared immediately before use
4 Light intensity is a measure of the rate of catalysis by the luciferases and is therefore temperature sensitive The
temperature optimum for the activity of both luciferases is approximately room temperature (20–25°C) so it is important
that the reagents be equilibrated to room temperature before beginning measurements To avoid the need to temperature
equilibrate reagents before use store the DualGlo® Luciferase Buff er and the DualGlo® Stop & Glo® Buff er at room
temperature If reagents are colder than room temperature place them in a room temperature water bath to equilibrate
before use
5 To achieve maximum reproducibility equilibrate cells in media to room temperature before performing
luciferase measurements
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3C Assay Procedure
1 Remove multiwell plates containing mammalian cells from the incubator Make certain that the plates are
compatible with the type of luminometer being used
2 Measuring fi refl y luciferase activity Add a volume of DualGlo® Luciferase Reagent equal to the culture
medium volume to each well and mix For 96well plates typically 75µl of reagent is added to cells grown in 75µl
of medium For 384well plates typically 20µl of reagent is added to cells grown in 20µl of medium
3 Wait at least 10 minutes then measure the fi refl y luminescence (consult your luminometer manual for proper use
of the instrument) Optimal results will be generated if the luminescence is measured within 2 hours of addition
of DualGlo® Luciferase Reagent
4 Measuring Renilla luciferase activity Add a volume of DualGlo® Stop & Glo® Reagent equal to the original
culture medium volume to each well and mix As noted in Step 2 this volume is typically 75µl for 96well plates
and 20µl for 384well plates
Note DualGlo® Stop & Glo® Reagent should be added to plate wells within 4 hours of addition of DualGlo®
Luciferase Reagent
5 Wait at least 10 minutes then measure luminescence Renilla luminescence should be measured in the same
plate order as the fi refl y luminescence was measured (Step 3) Optimal results will be generated if the
luminescence is measured within 2 hours of addition of DualGlo® Stop & Glo® Reagent
6 Calculate the ratio of luminescence from the experimental reporter to luminescence from the control reporter
Normalize this ratio to the ratio of a control well or series of control wells that are treated consistently on all
plates (see Section 6C) This normalization provides optimal and consistent results from the DualGlo®
Luciferase Assay System Relative Response Ratios can then be calculated from the Normalized Ratios See
Section 6C for more information and sample calculations
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4 Related Products
pGL4 Luciferase Reporter Vectors
Please visit wwwpromegacom to see a complete listing of our reporter vectors
Vector
Multiple
Cloning
Region
Reporter
Gene
Protein
Degradation
Sequence
Reporter
Gene
Promoter
Mammalian
Selectable
Marker Cat#
pGL410[luc2]Yesluc2A No No No E6651
pGL411[luc2P] Yes hPEST No No E6661
pGL412[luc2CP] Yes hCL1hPEST No No E6671
pGL413[luc2SV40] No No SV40 No E6681
pGL414[luc2Hygro] Yes No No Hygro E6691
pGL415[luc2PHygro] Yes hPEST No Hygro E6701
pGL416[luc2CPHygro] Yes hCL1hPEST No Hygro E6711
pGL417[luc2Neo] Yes No No Neo E6721
pGL418[luc2PNeo] Yes hPEST No Neo E6731
pGL419[luc2CPNeo] Yes hCL1hPEST No Neo E6741
pGL420[luc2Puro] Yes No No Puro E6751
pGL421[luc2PPuro] Yes hPEST No Puro E6761
pGL422[luc2CPPuro] Yes hCL1hPEST No Puro E6771
pGL423[luc2minP] Yes No minP No E8411
pGL424[luc2PminP] Yes hPEST No E8421
pGL425[luc2CPminP] Yes hCL1PEST No E8431
pGL426[luc2minPHygro] Yes No Hygro E8441
pGL427[luc2PminPHygro] Yes hPEST Hygro E8451
pGL428[luc2CPminPHygro] Yes hCL1PEST Hygro E8461
pGL429[luc2PCREHygro] No hPEST CRE Hygro E8471
pGL430[luc2PNFATREHygro] No hPEST NFATRE Hygro E8481
pGL431[luc2PGAL4UASHygro] No hPEST GAL4UAS Hygro C9351
pGL432[luc2PNFBREHygro] No hPEST NFBRE Hygro E8491
pGL433[luc2PSREHygro] No hPEST SRE Hygro E1340
pGL434[luc2PSRFREHygro] No hPEST SRFRE Hygro E1350
pGL436[luc2PMMTVHygro] No hPEST MMTV Hygro E1360
pGL450[luc2CMVHygro] No No CMV Hygro E1310
pGL451[luc2CMVNeo] No No CMV Neo E1320
Aluc2 synthetic fi refl y luciferase gene BhRluc synthetic Renilla luciferase gene
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pGL4 Luciferase Reporter Vectors (continued)
Vector
Multiple
Cloning
Region
Reporter
Gene
Protein
Degradation
Sequence
Reporter
Gene
Promoter
Mammalian
Selectable
Marker Cat#
pGL470[hRluc]YeshRlucB No No No E6881
pGL471[hRlucP] Yes hPEST No No E6891
pGL472[hRlucCP] Yes hCL1hPEST No No E6901
pGL473[hRlucSV40] No No SV40 No E6911
pGL474[hRlucTK] No No HSVTK No E6921
pGL475[hRlucCMV] No No CMV No E6931
pGL476[hRlucHygro] Yes No No Hygro E6941
pGL477[hRlucPHygro] Yes hPEST No Hygro E6951
pGL478[hRlucCPHygro] Yes hCL1hPEST No Hygro E6961
pGL479[hRlucNeo] Yes No No Neo E6971
pGL480[hRlucPNeo] Yes hPEST No Neo E6981
pGL481[hRlucCPNeo] Yes hCL1hPEST No Neo E6991
pGL482[hRlucPuro] Yes No No Puro E7501
pGL483[hRlucPPuro] Yes hPEST No Puro E7511
pGL484[hRlucCPPuro] Yes hCL1hPEST No Puro E7521
Aluc2 synthetic fi refl y luciferase gene BhRluc synthetic Renilla luciferase gene
Luciferase Assay Systems
Product Size Cat#
SteadyGlo® Luciferase Assay System* 100ml E2520
BrightGlo™ Luciferase Assay System* 100ml E2620
DualLuciferase® Reporter Assay System* 100 assays E1910
Luciferase Assay System* 100 assays E1500
Luciferase Assay Reagent 1000 assays E1483
Renilla Luciferase Assay System* 100 assays E2810
QuantiLum® Recombinant Luciferase* 1mg E1701
EnduRen™ Live Cell Substrate* 034mg E6481
ViviRen™ Live Cell Substrate* 037mg E6491
*Additional Sizes Available
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4 Related Products (continued)
Transfection Reagent
Product Size Cat#
FuGENE® HD Transfection Reagent 1ml E2311
5 × 1ml E2312
Luminometers
Product Size Cat#
GloMax®Multi+ Detection System with Instinct™ Software Base Instrument with Shaking each E8032
GloMax®Multi+ Detection System with Instinct™ Software
Base Instrument with Heating and Shaking each E9032
GloMax® 2020 Luminometry System each E5311
GloMax® 2020 Luminometry System with Single AutoInjector each E5321
GloMax® 2020 Luminometry System with Dual AutoInjector each E5331
GloMax® 96 Microplate Luminometer each E6501
GloMax® 96 Microplate Luminometer with Single Injector each E6511
GloMax® 96 Microplate Luminometer with Dual Injector each E6521
Plasmid DNA Purifi cation System
Product Size Cat#
PureYield™ Plasmid Midiprep System 25 preps A2492
100 preps A2495
300 preps A2496
PureYield™ Plasmid Maxiprep System 10 preps A2392
25 preps A2393
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5 References
1 Alam J and Cook JL (1990) Reporter genes Application to the study of mammalian gene transcription Anal
Biochem 188 245–54
2 Wood KV (1991) In Bioluminescence and Chemiluminescence Current Status Stanley P and Kricka L eds John
Wiley and Sons Chichester NY 543
3 Wood KV et al (1984) Synthesis of active fi refl y luciferase by in vitro translation of RNA obtained from adult
lanterns Biochem Biophys Res Comm 124 592–6
4 deWet JR et al (1985) Cloning of fi refl y luciferase cDNA and the expression of active luciferase in Escherichia
coli Proc Natl Acad SciUSA 82 7870–3
5 Matthews JC et al (1977) Purifi cation and properties of Renilla reniformis luciferase Biochemistry 16 85–91
6 Appendix
6A Overview of the DualGlo® Luciferase Assay System
Transcriptional regulation coupled to reporter gene expression is routinely used to study a wide range of physiological
responses A common example is the analysis of receptor function by quantitating the action of specifi c reporter response
elements on gene expression Firefl y luciferase has been used as a simple convenient and sensitive reporter gene for this
type of analysis
An increase in the transcription or translation of a reporter molecule like fi refl y luciferase is easily tracked in a biological
system Firefl y luciferase is immediately functional upon translation Thus if the amount of luminescence from an
experimental sample is greater than the luminescence from a control sample an increase in transcription or translation
has occurred
Studies of antagonists or other factors that decrease transcription or translation are diffi cult to interpret when using a
single reporter gene A decrease in an experimental reporter response (fi refl y luciferase in this case) can be caused by a
specifi c eff ect on the reporter or by a global eff ect such as cell death Normalization of an experimental reporter with a
control reporter from the same sample such as Renilla luciferase allows a distinction to be made between specifi c and
global eff ects A decrease in fi refl y luminescence with Renilla luminescence remaining unchanged indicates a specifi c
impact of the experimental condition A decrease of both luminescences indicates a global impact on the cell or cell
population (ie cell death inhibition of cell growth variable initial cell numbers)
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6A Overview of the DualGlo® Luciferase Assay System (continued)
The DualLuciferase® Reporter Assay System (DLR™ Assay System Cat# E1910) allows measurement of both fi refl y
and Renilla luciferases from a single sample This assay system however generates luminescence that rapidly
decreases in intensity The fi refl y luciferase signal decreases 50 in approximately 12–15 minutes and the Renilla
luciferase signal decreases 50 in less than 3 minutes These signal kinetics make the measurement of fi refl y and
Renilla luciferases diffi cult if large numbers of samples are to be measured in 96 or 384well plates Although robotic
systems can easily add reagents to all the samples on a plate at the same time the signals are too unstable to easily
measure the luminescence from the entire plate without having to calculate time corrections
The DualGlo® Luciferase Assay System with its stabilized luminescent signals and halflife of approximately 2 hours
easily can be used to measure both fi refl y and Renilla luminescence in multiwell plates Many plates containing
experimental samples can be batch processed all plates can be fi lled with reagent at one time and the luminescence
from each plate can be measured in series In this way the DualGlo® Luciferase Assay System can easily and rapidly
quantitate both fi refl y and Renilla luciferases allowing one to distinguish between global and specifi c eff ects
6B Development of the Assay
Firefl y and Renilla luciferases have distinct evolutionary origins and have very diff erent enzyme structures and
substrates Promega has been able to exploit these diff erences so that the two luciferases can be measured in
succession with fi refl y luciferase luminescence elicited by one reagent while a second reagent simultaneously
quenches the fi refl y luciferase and elicits Renilla luciferase luminescence
Firefl y and Renilla luciferases do not require posttranslational modifi cation and thus are fully functional enzymes
immediately after translation (34) In order to generate luminescence fi refl y luciferase requires beetle luciferin ATP
magnesium and molecular oxygen Renilla luciferase requires only coelenterate luciferin (coelenterazine) and molecular
oxygen (Figure 2) Firefl y and Renilla luciferases undergo spontaneous inactivation after generating luminescence This
inactivation causes the fl ashtype kinetics seen in assays that have been optimized for maximal assay sensitivity like
the DLR™ Assay System (Figure 3) In order to generate luminescence with signal stability that is amenable to robotic
measurements the rate of inactivation and subsequently the rate of catalysis must be slowed For this reason glotype
reagents developed for use in highthroughput screening like the SteadyGlo® and DualGlo® Reagents have lower
luminescences than fl ashtype reagents that have been developed for maximal sensitivity like the Luciferase and
DLR™ Assay Reagents
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1399MA03_6A
HO S
N S
N O S
N S
N
OCOOH
+ATP+O2
Recombinant Firefly
Luciferase
+AMP+PPi+CO2+Light
Beetle Luciferin Oxyluciferin
Mg2+
N
H
N N
O OH
+O2
HO
N
N NH
O
OH
+CO2+Light
HO
Renilla
Luciferase
Coelenterazine Coelenteramide
Figure 2 Bioluminescent reactions catalyzed by fi refl y and Renilla luciferases Monooxygenation of beetle
luciferin is catalyzed by fi refl y luciferase in the presence of Mg2+ ATP and molecular oxygen Unlike beetle luciferin
coelenterazine undergoes monooxygenation catalyzed by Renilla luciferase but requires only molecular oxygen
Fi
r
efly Luminescence (RLU)
Time (Minutes)
10
0 20 40 60 80 100 120
100
1000
10000
Renilla
Luminescence (RLU)
Time (Minutes)
10
0 20 40 60 80 100 120
100
1000
10000
3619MA02_2A
A B
DLR™
DualGlo™
Figure 3 Comparison of the signal decay of luciferases in DualGlo® Reagent and DLR™ Assay Reagent
The two luciferase assay reagents were compared using 167 × 10–9M (plus 1mgml gelatin) firefly and Renilla
luciferase (20µl and 100µl of enzyme mix were used with DLR™ and DualGlo® Reagents respectively) The
luciferases were diluted in RPMI 1640 before assaying with DualGlo® Luciferase Reagent and in Passive Lysis Buffer
(Cat# E1941) before assaying with DLR™ Assay Reagent The DLR™ Assay measurements were taken immediately
after reagent addition while the DualGlo® measurements were taken after a 10minute incubation at room
temperature Luminescence was integrated over 05 seconds per well at regular intervals until 2 hours after reagent
addition Panel A Firefly luciferase activity in DualGlo® and DLR™ Assays Panel B Renilla luciferase activity in
DualGlo® and DLR™ Assays
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6B Development of the Assay (continued)
Quenching Eff ect
Providing the ability to measure fi refl y luminescence is only one of the requirements for the DualGlo® Luciferase
Assay System After measurement of the fi refl y luminescence the DualGlo® Stop & Glo® Reagent must simultaneously
quench fi refl y luminescence and generate Renilla luminescence
The DualGlo® Stop & Glo® Reagent has been designed to decrease the luminescence of the fi refl y luciferase reaction
by at least 10000fold (Figure 4) Thus if the fi refl y luciferase luminescence generated 530000 Relative Light Units
per second the maximum fi refl y luminescence measured 10 minutes after the DualGlo® Stop & Glo® Reagent addition
would be 53 Relative Light Units per second The promoter for the control reporter (either fi refl y or Renilla luciferase)
should be chosen so that the luminescence from the fi refl y luciferase is no more than 100 times the luminescence from
the Renilla luciferase If this is the case the fi refl y luciferase luminescence will contribute no more than 1 to the
Renilla luciferase luminescence and variability in the fi refl y luciferase signal will not noticeably aff ect the Renilla
luciferase signal
3620MA02_2A
Firefly
Luciferase
Activity
Quenched
Luminescence
Renilla
Luciferase
Activity
Luminescence (RLU)
10
100
1000
10000
100000
1000000
10000000
1900000
30
(60000fold
quench)
170000
Figure 4 Measurement of luciferase activities before and after addition of DualGlo® Stop & Glo®
Reagent Both fi refl y and Renilla luciferase reporter activities were quantitated using a 100µl sample of purifi ed
fi refl y or Renilla luciferase (167 × 10–9M and 167 × 10–10M respectively with 1mgml gelatin) The concentration
of Renilla luciferase is 10fold less than that of fi refl y luciferase To demonstrate the effi cient quenching of fi refl y
luciferase by DualGlo® Stop & Glo® Reagent an equal volume of DualGlo® Stop & Glo® Buff er (which does not
contain the substrate for Renilla luciferase) was added to the DualGlo® Luciferase Reagent with fi refl y luciferase
Firefl y luciferase luminescence was quenched by greater than 4 orders of magnitude with 00016 residual activity
RLU relative light units
The DualGlo® Stop & Glo® Reagent has been designed to provide simultaneous quenching of the fi refl y luciferase
signal and sustained luminescence for the Renilla luciferase reaction Renilla luciferase does not use any of the
substrates for the fi refl y reaction except molecular oxygen This permits the DualGlo® Stop & Glo® Reagent to both
quench the fi refl y reaction and generate the Renilla luciferase reaction Renilla luciferase is similar to fi refl y luciferase
in that the enzyme inactivates itself As with the fi refl y luciferase the rate of catalysis must be slowed if the luminescent
signal is to be stabilized Glotype Renilla assays (DualGlo® Luciferase Assay) are therefore not as sensitive as
fl ashtype assays (DLR™ and Renilla Luciferase Assays)
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Unlike the fi refl y luciferase substrate (beetle luciferin) coelenterazine will autooxidize in the absence of Renilla
luciferase This enzymeindependent reaction is termed autoluminescence Reaction conditions such as high detergent
levels created in homogeneous assays can increase the level of autoluminescence which can limit the sensitivity of the
Renilla luciferase reporter system The DualGlo® Luciferase Assay System uses unique technology that reduces
autoluminescence to levels that are often undetectable thereby increasing the sensitivity of the Renilla luciferase
reagent of the DualGlo® Luciferase Assay System by >100fold over nonoptimized reagents (Figure 5)
3621MA02_2A
Renilla
Lucife
r
ase
Luminescence (RLU)
[Renilla Luciferase] (M)
Optimized Reagent
Nonoptimized Reagent
Optimized Autoluminescence
Nonoptimized Autoluminescence
01
10–19 10–18 10–17 10–16 10–15 10–14 10–13 10–12
1
10
100
1000
10000
100000
1000000
Figure 5 The DualGlo® Luciferase Assay System is optimized for minimal autoluminescence from
Renilla luciferase substrate Luminescence from a titration of Renilla luciferase was compared in DualGlo®
Reagent before and after optimization for homogeneous assay formats Luciferase concentrations varied over a titration
range of 1 × 10–12 to 1 × 10–19 molesreaction One hundred microliters of Renilla luciferase in RPMI 1640 (containing
1mgml gelatin) was added to 100µl of DualGlo® Luciferase Reagent and 100µl of DualGlo® Stop & Glo® Reagent
either optimized or nonoptimized for homogeneous assay format Samples were incubated for 10 minutes at 22°C then
measured on a Turner Designs Model 20e luminometer Light emission was integrated over 5 seconds after an initial
2second preread delay Limit of detection values shown (horizontal lines labeled Optimized and Nonoptimized)
represent background plus three standard deviations and were determined for each assay by performing the assay
without enzyme
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6C Data Analysis
Background Subtraction
For maximal accuracy the luminescence measurements of both fi refl y and Renilla luciferases should be background
subtracted Neither enzyme is endogenously expressed in mammalian cells so the source of background luminescence
is either a characteristic of the luminometer or of the luminescent substrate Beetle luciferin one of the fi refl y luciferase
substrates does not generate light in the absence of luciferase in the DualGlo® Reagents A proprietary chemistry
minimizes the enzymeindependent luminescence (autoluminescence) exhibited by coelenterate luciferin the substrate
for Renilla luciferase in the DualGlo® Stop & Glo® Reagent Background luminescence for both luciferase reagents
therefore may not be measurable above the background on many luminometers
Background measurements for fi refl y luciferase should be taken from samples consisting of nontransfected cells and
DualGlo® Luciferase Reagent For Renilla luciferase background measurements should be taken from samples
containing nontransfected cells and both DualGlo® Luciferase Reagent and DualGlo® Stop & Glo® Reagent Sample
volumes for background measurements must be the same as experimental sample volumes and contain the same
mediasera combinations as the experimental samples
Normalizing Ratios
Normalizing the results from each experimental sample to control samples repeated on each plate minimizes the impact
of variables like temperature plate order and timing on the ratio of experimentalcontrol reporter activity
Example 1
An experiment includes 20 plates If the timing of measurement of the plates is changed between the fi refl y and Renilla
luminescence measurements then the ratio of fi refl y luminescence to Renilla luminescence will be diff erent on each of the
plates because the two reporter signals would have decayed by diff erent amounts (changing the order of the plates changes
the length of time between reagent addition and plate reading) For instance the luminometer jammed 20 minutes after
the DualGlo® Stop & Glo® Reagent was added to a large stack of plates
Ratio of plate 1 control fi refl y luminescenceRenilla luminescence 17
Ratio of plate 1 well A1 fi refl y luminescenceRenilla luminescence 34
Both fi refl y luciferase and Renilla luciferase were measured 10 minutes after the reagents were added
Relative ratios are control 1717 1 and well A1 3417 2
However for plate 12 the fi refl y luciferase was measured 40 minutes after DualGlo® Luciferase Reagent was added
but because of the luminometer jam the Renilla luciferase was not measured until 75 minutes after the DualGlo®
Stop & Glo® Reagent was added
Ratio of plate 12 control fi refl y luminescenceRenilla luminescence 21
Ratio of plate 12 well A1 fi refl y luminescenceRenilla luminescence 42
Relative ratios are control 2121 1 and well A1 4221 2
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Example 2
The DualGlo® Assay is being performed in a cell culture medium (MEM) that causes the fi refl y and Renilla
luciferases to decay at diff erent rates (fi refl y luciferase retains ~90 activity over 2 hours Renilla luciferase retains
~75 activity over 2 hours) Normalizing the ratios permits comparison of these ratios between plates in the same
experiment
Ratio of plate 1 control fi refl y luminescenceRenilla luminescence 64
Ratio of plate 1 well A1 fi refl y luminescenceRenilla luminescence 16
Both fi refl y luciferase and Renilla luciferase were measured 10 minutes after the reagents were added
Relative ratios were control 6464 1 and well A1 1664 025
For plate 12 both fi refl y luciferase and Renilla luciferase were measured 65 minutes after the reagents were added
but the two signals do not decay at the same rate Thus the raw ratio changes
Ratio of plate 12 control fi refl y luminescenceRenilla luminescence 69
Ratio of plate 12 well A1 fi refl y luminescenceRenilla luminescence 17
Relative ratios are control 6969 1 and well A1 1769 025
Other factors may aff ect the ratio of experimentalcontrol reporter As seen in Section 6D and mentioned above
chemical components of the mediumserum combination aff ect the fi refl y luciferase signal stability diff erently than
they aff ect the Renilla luciferase signal stability This is also true of changes in temperature and other parameters that
aff ect enzyme reactions Normalization of the experimentalcontrol reporter activity is essential for easy comparison
of samples with minimal variability across an experiment
Relative Response Ratios
A Relative Response Ratio (RRR) can be determined to assist in quantitation of the impact of an experimental treatment
on reporter gene expression The RRR permits the comparison of multiple treatments from diff erent experiments because
it provides a framework within which the eff ect of the treatment can be placed
Calculation of RRR requires the inclusion of 2 sets of controls on each plate a positive control that provides maximal
luminescence and a negative control that provides minimal luminescence For an experiment that monitors down
regulation of the experimental reporter by chemical treatments the positive control for the experimental series might
be no treatment The negative control would be treatment with some compound that had previously been shown to
drastically inhibit the experimental reporter If these 2 samples are included on every plate the impact of each new
compound can be quantitatively evaluated by its infl uence on the experimental reporter within the context of the positive
and negative control
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6C Data Analysis (continued)
Example
If the ratio of experimental reporter luminescencecontrol reporter luminescence is 53 for the positive control 13 for the
negative control and 22 for the experimental treatment all of these values can be scaled so that the positive control is
assigned the value of 1 and the negative control is assigned the value of 0 The RRR for each experimental treatment can
then be calculated using this formula
RRR (experimental sample ratio) – (negative control ratio)
(positive control ratio) – (negative control ratio)
The RRR for the experimental treatment example listed above would be
RRR 22 – 13 040 or 40 53 – 13
The experimental compound is 40 as eff ective as the negative control at decreasing expression of the experimental
reporter at this concentration A hierarchy of effi cacy for the experimental system would be i) no treatment generated
a RRR of 100 ii) the experimental compound generated a RRR of 40 and iii) the negative control generated a RRR
of 0 Those compounds that are most signifi cant will have RRRs that are negative since they would be more eff ective
inhibitors than the negative control
Note If the absolute luminescence values are close to background the luminescences will need to be background subtracted
before the ratios are calculated
6D Conditions Aff ecting Assay Performance
The data presented in this section are intended to provide a general overview of assay characteristics under a wide
range of experimental conditions The cell culture medium accounts for 50 of the fi refl y luciferase and 33 of the
Renilla luciferase reaction volumes Thus medium can aff ect assay performance The chemistry used for the DualGlo®
Luciferase Assay System is unique when compared to other assays developed for highthroughput analysis and
compared to the DLR™ Assay System Therefore the data presented in this technical manual may not be applicable to
other luciferase assay systems
Purifi ed fi refl y and Renilla luciferases were diluted into culture medium to generate the data presented in this section
This was done to illustrate the performance characteristics without adding the experimental complexities common to cell
culture However as shown in Figure 6 purifi ed luciferases diluted into culture medium show little or no diff erence
when compared to enzymes expressed in transfected cells Gelatin (1mgml) was added to the wells to simulate the
protein that would normally be contributed by cells Addition of gelatin is not required when using the DualGlo®
Luciferase Assay System with cells
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3622MA02_2A
A B
C D
0
0 20 40 60 80 100 120
20
40
60
80
100
Rela
t
ive Fi
r
efly Luminescence
(Pe
r
cen
t
Remaining Af
t
e
r
10 Minu
t
es)
Time (Minutes)
0
0 20 40 60 80 100 120
20
40
60
80
100
Rela
t
ive
Renilla
Luminescence
(Pe
r
cen
t
Remaining Af
t
e
r
10 Minu
t
es)
Time (Minutes)
0
0 20 40 60 80 100 120
20
40
60
80
100
Rela
t
ive
Renilla
Luminescence
(Pe
r
cen
t
Remaining Af
t
e
r
10 Minu
t
es)
Time (Minutes)
0
0 20 40 60 80 100 120
20
40
60
80
100
Rela
t
ive Fi
r
efly Luminescence
(Pe
r
cen
t
Remaining Af
t
e
r
10 Minu
t
es)
Time (Minutes)
CHO Lysate
Purified Luciferase
CHO Lysate
Purified Luciferase
293 Lysate
Purified Luciferase
293 Lysate
Purified Luciferase
Figure 6 Reaction kinetics for purified firefly and Renilla luciferase and for luciferases expressed by
transiently transfected mammalian cells Samples in 96well plates consisted of 75µl of either purified firefly and
Renilla luciferases (both at 167 × 10–9M 1mgml gelatin as a protein carrier) or mammalian cells transfected with firefly
and Renilla luciferase genes (using pCI Mammalian Expression Vector [Cat# E1731] with luc+ inserted at XbaIXhoI sites
for firefly luciferase and the phRLSV40 Vector for Renilla luciferase [Cat# E6261]) in the same growth medium Seventy
five microliters of DualGlo® Luciferase Reagent was added to plate wells and mixed on a plate shaker After a 10minute
room temperature incubation luminescence was integrated over 05 seconds per well periodically for 2 hours after reagent
addition Stop & Glo® Reagent was then added to wells and mixed on a plate shaker and luminescence was integrated as
noted above Firefly luciferase was measured in CHO cells in F12 medium (Panel A) and in 293 cells in DMEM (Panel B)
Renilla luciferase was measured in CHO cells in F12 medium (Panel C) and in 293 cells in DMEM (Panel D) As these data
show very little difference in relative luminescence is apparent over time between transfected cells and purified enzyme in the
same culture medium Number of samples 6 for cell lysate data 3 for purified enzyme data
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6D Conditions Aff ecting Assay Performance (continued)
Culture Medium
When performing the DualGlo® Luciferase Assay the culture medium and any compounds added to the medium make
up half of the chemical environment of the fi refl y luciferase reaction and onethird of the environment of the Renilla
luciferase reaction Although the DualGlo® Reagents are designed to work with many common culture media
compositional diff erences between the diff erent media may aff ect the assay characteristics (ie light intensity and
signal stability)
The DualGlo® Luciferase Reagents were designed to provide relatively high luminescence with a halflife of approxi
mately 2 hours when used with common growth medium However performance diff erences are evident between these
media (Figure 7) as well as between the same media from diff erent manufacturers Although these diff erences are
generally small and do not diminish the utility of the DualGlo® Luciferase Assay System controls should be incorpo
rated into every batch of plates to correct for this variability
3623MA02_2A
DMEM F12 MEMα RPMI
Rela
t
ive Luminescence (Pe
r
cen
t
)
Firefly
Renilla
Signal S
t
abili
t
y (Pe
r
cen
t
Luminescence
Remaining Af
t
e
r
120 Minu
t
es)
0
20
40
60
80
100
DMEM F12 MEMα RPMI
0
20
40
60
80
100
A B
Figure 7 Relative luminescence intensity and signal stability of fi refl y and Renilla luciferase in four
common media Purifi ed fi refl y or Renilla enzyme (167 × 10–9M 1mgml gelatin) was added to a 96well plate at
100µl per well Dilutions were made in RPMI 1640 DMEM MEM or F12 medium Either DualGlo® Luciferase
Reagent (for fi refl y luciferase) or DualGlo® Luciferase Reagent plus DualGlo® Stop & Glo® Reagent (for Renilla
luciferase) was added and luminescence measurements were integrated over 05 seconds per well Panel A
Luminescence measurements were taken 10 minutes after reagent addition Luminescence is shown relative to the
light output generated in RPMI 1640 for both fi refl y and Renilla luciferases Panel B Signal stability in various media
expressed as percent luminescence remaining after 2 hours Number of samples 3 relative standard error 34
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Serum
The DualGlo® Reagents are compatible with medium containing serum The reagents have been designed for use with
serum concentrations from 0–10 and the luminescent signals generated are minimally aff ected by the presence of
fetal bovine or calf serum (Figure 8)
3624MA02_2A
C D
Signal S
t
abili
t
y (Pe
r
cen
t
Luminescence
Remaining Af
t
e
r
120 Minu
t
es)
0
0246810
20
40
60
80
100
120
Rela
t
ive Luminescence (Pe
r
cen
t
)
[Serum] (Percent of Medium)
0
0246810
20
40
60
80
100
120
Rela
t
ive Luminescence (Pe
r
cen
t
)
[Serum] (Percent of Medium)
0
0246810
20
40
60
80
100
120
[Serum] (Percent of Medium)
Signal S
t
abili
t
y (Pe
r
cen
t
Luminescence
Remaining Af
t
e
r
120 Minu
t
es)
0
0246810
20
40
60
80
100
120
[Serum] (Percent of Medium)
CS
FBS
CS
FBS
Figure 8 Eff ects of serum on luminescence intensity and signal stability Purifi ed fi refl y or Renilla luciferase
(167 × 10–9M 1mgml gelatin) was added to a 96well plate at 100µl per well F12 medium containing various
concentrations of either fetal bovine serum (FBS) or calf serum (CS) was used for the assay Either DualGlo® Luciferase
Reagent (for fi refl y luciferase) or DualGlo® Luciferase Reagent plus DualGlo® Stop & Glo® Reagent (for Renilla
luciferase) was added and luminescence measurements were integrated over 05 seconds per well Firefl y luciferase
relative luminescence (Panel A) and signal stability (Panel B) were measured in medium containing fetal bovine
serum (FBS) and calf serum (CS) Renilla luciferase relative luminescence (Panel C) and signal stability (Panel D)
were measured in medium containing various concentrations of FBS and CS Luminescence was compared for samples
containing serum and for samples containing no serum Signal stability expressed as percent of luminescence remaining
after 2 hours Number of samples 3 relative standard error 39
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6D Conditions Aff ecting Assay Performance (continued)
Phenol Red
Phenol red is a pH indicator commonly used in cell culture media Many commercial media formulations contain
5–15mgL phenol red causing the characteristic red color This compound can reduce assay sensitivity (Figure 9)
However in most applications the presence of phenol red will not signifi cantly aff ect the utility of the DualGlo®
Luciferase Assay System To minimize its eff ect use as little phenol red as possible in culture medium
3625MA02_2A
A B
Rela
t
ive Luminescence (Pe
r
cen
t
)
[Phenol Red] (mgL)
Firefly Luciferase
Renilla Luciferase
Signal S
t
abili
t
y (Pe
r
cen
t
Luminescence
Remaining Af
t
e
r
120 Minu
t
es)
0
01020
20
40
60
80
100
[Phenol Red] (mgL)
0
01020
20
40
60
80
100
Figure 9 The eff ect of phenol red on luminescence intensity and signal stability Purifi ed fi refl y or Renilla
luciferase (167 × 10–9M 1mgml gelatin) was added to a 96well plate at 100µl per well MEM containing various
concentrations of phenol red was used for the assay Either DualGlo® Luciferase Reagent (for fi refl y luciferase) or DualGlo®
Luciferase Reagent plus DualGlo® Stop & Glo® Reagent (for Renilla luciferase) was added and luminescence measurements
were integrated over 05 seconds per well Luminescence is compared for samples containing phenol red and for samples
containing no phenol red Panel A Luminescence is shown relative to that measured without phenol red Panel B Signal
stability in various concentrations of phenol red expressed as percent of luminescence remaining after 2 hours Number of
samples 3 relative standard error 40
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Organic Solvents
Organic solvents are typically present in reporter gene assays because they are used to stabilize and solubilize
screening compounds DMSO ethanol and methanol have little eff ect on the assay (Figure 10) The compatibility of
other solvents should be verifi ed prior to use
3626MA02_2A
C D
0
0123
20
40
60
80
100
120
Rela
t
ive Luminescence (Pe
r
cen
t
)
[Solvent] (Percent of Media)
0
0123
20
40
60
80
100
120
Rela
t
ive Luminescence (Pe
r
cen
t
)
[Solvent] (Percent of Media)
0
0123
20
40
60
80
100
120
[Solvent] (Percent of Media)
DMSO
Methanol
Ethanol
DMSO
Methanol
Ethanol
Signal S
t
abili
t
y (Pe
r
cen
t
Luminescence
Remaining Af
t
e
r
120 Minu
t
es)
0
0123
20
40
60
80
100
120
[Solvent] (Percent of Media)
Signal S
t
abili
t
y (Pe
r
cen
t
Luminescence
Remaining Af
t
e
r
120 Minu
t
es)
Figure 10 The eff ect of organic solvents on luminescence intensity and signal stability Purifi ed fi refl y or
Renilla luciferase (167 × 10–9M 1mgml gelatin) was added to a 96well plate at 100µl per well F12 medium
containing various concentrations of DMSO methanol or ethanol was used for the assay Either DualGlo® Luciferase
Reagent (for fi refl y luciferase) or DualGlo® Luciferase Reagent plus DualGlo® Stop & Glo® Reagent (for Renilla
luciferase) was added and luminescence measurements were integrated over 05 seconds per well Firefl y luciferase
relative luminescence (Panel A) and signal stability (Panel B) as well as Renilla luciferase relative luminescence
(Panel C) and signal stability (Panel D) were determined in various concentrations of solvent Luminescence is
expressed relative to samples without organic solvents Signal stability is expressed as percent of luminescence
remaining after 2 hours Number of samples 3 relative standard error 40
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6D Conditions Aff ecting Assay Performance (continued)
Temperature
Both fi refl y and Renilla luciferases are temperature sensitive thus temperature is an important factor in experimental
precision (Figure 11) Good precision can be achieved most easily by performing all experiments at room temperature
which is near the temperature optima for both fi refl y and Renilla luciferases The assay reagents should be at room
temperature before beginning measurements
As mentioned previously the DualGlo® Buff ers can be stored at room temperature to avoid the need for temperature
equilibration before use The heat capacity of the substrates is low therefore reconstitution of the DualGlo®
Substrates with the room temperature DualGlo® Buff ers produces reagents that are ready for use If temperature
equilibration is needed incubate reagents in a water bath at room temperature Do not use a water bath set higher
than 25°C
Lower temperatures result in increased signal stabilities but lower luminescent intensities If cold reagent is used the
luminescence will slowly increase during the experiment as the reagent warms High temperatures aff ect fi refl y and
Renilla luciferases diff erently however both signals become less stable This can occur if the culture plates are too
warm or if the luminometer produces excess heat within the reading chamber As shown in Figure 11 fi refl y luciferase
is more sensitive to changes in temperature than Renilla luciferase
3627MA02_2A
A B
Signal S
t
abili
t
y (Pe
r
cen
t
Remaining
Af
t
e
r
120 Minu
t
es)
18 20 22 24 26Rela
t
ive Luminescence (Pe
r
cen
t
)
Temperature (°C)
Firefly Luciferase
Renilla Luciferase
0
20
40
60
80
100
18 20 22 24 26
Temperature (°C)
0
20
40
60
80
100
Figure 11 The eff ect of temperature on luciferase luminescence Purifi ed fi refl y or Renilla luciferase
(167 × 10–9M 1mgml gelatin) was added to a luminometer tube containing 100µl of DualGlo® Luciferase Reagent
(for fi refl y luciferase) or DualGlo® Luciferase Reagent plus DualGlo® Stop & Glo® Reagent (for Renilla luciferase)
Samples were incubated at various temperatures and light emission was measured on a Turner Designs Model 20e
luminometer integrated over 10 seconds after a 2second preread delay Panel A Luminescence at 10 minutes is
shown relative to that measured at 22°C Panel B Signal stability at various temperatures is expressed as percent of
luminescence remaining after 2 hours Number of samples 3 relative standard error 31
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(a)US Pat Nos 7078181 7108996 and 7118878 European Pat No 1297337 and other patents pending
(b) US Pat No 5744320 European Pat No 0 833 939 and Japanese Pat No 360160
(c)Certain applicati ons of this product may require licenses from others
© 2002 2005 2006 2009 2010 2011 2015 Promega Corporati on All Rights Reserved
DualGlo DualLuciferase GloMax Quanti Lum SteadyGlo and Stop & Glo are registered trademarks of Promega Corporati on BrightGlo DLR
EnduRen PureYield and ViviRen are trademarks of Promega Corporati on
FuGENE is a registered trademark of Fugent LLC USA
Products may be covered by pending or issued patents or may have certain limitati ons Please visit our Web site for more informati on
All prices and specifi cati ons are subject to change without prior noti ce
Product claims are subject to change Please contact Promega Technical Services or access the Promega online catalog for the most uptodate
informati on on Promega products
6E Summary of Changes
The following change was made to the 915 version of this document
Expired patent information was removed

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