Reporter Assay System Instructions for use of Products

TECHNICAL MANUAL
DualLuciferase®
Reporter Assay System
Instructions for use of Products
E1910 and E1960
Revised 615
TM040
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DualLuciferase®
Reporter Assay System
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
1 Description2
1A DualLuciferase® Reporter Assay Chemistry3
1B Format of the DualLuciferase® Reporter Assay5
1C Passive Lysis Buffer6
2 Product Components and Storage Conditions8
3 The pGL4 Luciferase Reporter Vectors9
3A Description of pGL4 Vectors9
3B Important Considerations for CoTransfection Experiments 9
4 Instrument Considerations10
4A SingleSample Luminometers10
4B MultiSample and PlateReading Luminometers10
4C Scintillation Counters11
5 Preparation of Cell Lysates Using Passive Lysis Buffer12
5A Passive Lysis Buffer Preparation12
5B Passive Lysis of Cells Cultured in Multiwell Plates12
5C Active Lysis of Cells by Scraping13
6 DualLuciferase® Reporter Assay Protocol14
6A Preparation of Luciferase Assay Reagent II14
6B Preparation of Stop & Glo® Reagent15
6C Standard Protocol15
6D Important Considerations for Cleaning Reagent Injectors18
6E Determination of Assay Backgrounds19
7 References21
8 Appendix22
8A Composition of Buffers and Solutions22
8B Related Products22
9 Summary of Changes25
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1 Description
Genetic reporter systems are widely used to study eukaryotic gene expression and cellular physiology Applications
include the study of receptor activity transcription factors intracellular signaling mRNA processing and protein
folding Dual reporters are commonly used to improve experimental accuracy The term dual reporter refers to the
simultaneous expression and measurement of two individual reporter enzymes within a single system Typically the
experimental reporter is correlated with the effect of specific experimental conditions while the activity of the
cotransfected control reporter provides an internal control that serves as the baseline response Normalizing the
activity of the experimental reporter to the activity of the internal control minimizes experimental variability caused by
differences in cell viability or transfection efficiency Other sources of variability such as differences in pipetting
volumes cell lysis efficiency and assay efficiency can be effectively eliminated Thus dualreporter assays often allow
more reliable interpretation of the experimental data by reducing extraneous influences
The DualLuciferase® Reporter (DLR™) Assay System(a–c) provides an efficient means of performing dualreporter
assays In the DLR™ Assay the activities of firefly (Photinus pyralis) and Renilla (Renilla reniformis also known as
sea pansy) luciferases are measured sequentially from a single sample The firefly luciferase reporter is measured first
by adding Luciferase Assay Reagent II (LAR II) to generate a stabilized luminescent signal After quantifying the firefly
luminescence this reaction is quenched and the Renilla luciferase reaction is simultaneously initiated by adding
Stop & Glo® Reagent to the same tube The Stop & Glo® Reagent also produces a stabilized signal from the Renilla
luciferase which decays slowly over the course of the measurement In the DLR™ Assay System both reporters yield
linear assays with subattomole sensitivities and no endogenous activity of either reporter in the experimental host cells
Furthermore the integrated format of the DLR™ Assay provides rapid quantitation of both reporters either in
transfected cells or in cellfree transcriptiontranslation reactions
Promega offers the pGL4 series of firefly and Renilla luciferase vectors designed for use with the DLR™ Assay Systems
These vectors may be used to cotransfect mammalian cells with experimental and control reporter genes
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1A DualLuciferase® Reporter Assay Chemistry
Firefly and Renilla luciferases because of their distinct evolutionary origins have dissimilar enzyme structures and
substrate requirements These differences make it possible to selectively discriminate between their respective
bioluminescent reactions Thus using the DLR™ Assay System the luminescence from the firefly luciferase reaction
may be quenched while simultaneously activating the luminescent reaction of Renilla luciferase
Firefly luciferase is a 61kDa monomeric protein that does not require posttranslational processing for enzymatic activity
(12) Thus it functions as a genetic reporter immediately upon translation Photon emission is achieved through oxidation of
beetle luciferin in a reaction that requires ATP Mg2+ and O2 (Figure 1) Under conventional reaction conditions the oxidation
occurs through a luciferylAMP intermediate that turns over very slowly As a result this assay chemistry generates a flash
of light that rapidly decays after the substrate and enzyme are mixed
Many of our Luciferase Assay Reagents for quantitating firefly luciferase incorporate coenzyme A (CoA) to provide
more favorable overall reaction kinetics (3) In the presence of CoA the luciferase assay yields stabilized luminescence
signals with significantly greater intensities (Figure 2) than those obtained from the conventional assay chemistry The
firefly luciferase assay is extremely sensitive and extends over a linear range covering at least seven orders of magnitude
in enzyme concentration (Figure 3)
Renilla luciferase a 36kDa monomeric protein is composed of 3 carbohydrate when purified from its natural source
Renilla reniformis (4) However like firefly luciferase posttranslational modification is not required for its activity
and the enzyme may function as a genetic reporter immediately following translation The luminescent reaction
catalyzed by Renilla luciferase utilizes O2 and coelenterateluciferin (coelenterazine Figure 1)

Figure 1 Bioluminescent reactions catalyzed by firefly and Renilla luciferases
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1A DualLuciferase® Reporter Assay Chemistry (continued)
In the DLR™ Assay chemistry the kinetics of the Renilla luciferase reaction provide a stabilized luminescent signal that
decays slowly over the course of the measurement (Figure 2) Similar to firefly luciferase the luminescent reaction
catalyzed by Renilla luciferase also provides extreme sensitivity and a linear range generally extending six orders of
magnitude (Figure 3) Note that the effective range of the luminescent reactions may vary depending on the type of
luminometer (eg 96well versus singlesample) used
An inherent property of coelenterazine is that it emits lowlevel autoluminescence in aqueous solutions Originally this
drawback prevented sensitive determinations at the lower end of enzyme concentration Additionally some types of
nonionic detergents commonly used to prepare cell lysates (eg Triton® X100) greatly intensify coelenterazine
autoluminescence The DLR™ Assay Systems include proprietary chemistry that reduces autoluminescence to a level
that is not measurable for all but the most sensitive luminometers Passive Lysis Buffer is formulated to minimize the
effect of lysate composition on coelenterazine autoluminescence In addition the DLR™ Assay Systems include two
reconstituted assay reagents Luciferase Assay Reagent II and Stop & Glo® Reagent that combine to suppress
coelenterazine autoluminescence
0
10
20
30
40
50
60
70
80
90
100
0 24681012
Activity ( peak)
Time (sec)
Firefly
Renilla
Figure 2 Luminescent signals generated in the DualLuciferase® Reporter Assay System by firefly and
Renilla luciferases
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10042MA
Firefly luciferase
Renilla luciferase
r² 09996
r² 09993
1 × 103
1 × 104
1 × 105
1 × 106
1 × 107
1 × 108
1 × 109
1 × 1010
Luciferase Concentration (molesreaction)
1 × 10
–20
1 × 10
–19
1 × 10
–18
1 × 10
–17
1 × 10
–16
1 × 10
–15
1 × 10
–14
1 × 10
–13
1 × 10
–12
Figure 3 Comparison of the linear ranges of firefly and Renilla luciferases The DLR™ Assay was performed
with a mixture of purified firefly and Renilla luciferases prepared in PLB containing 1mgml BSA A Promega
GloMax® 2020 Luminometer was used to measure luminescence As shown in this graph with the DLR™ Assay
System the linear range of the firefly luciferase assay is eight orders of magnitude providing detection sensitivity
of ≤01 femtogram (approximately 10–21 mole) of firefly luciferase reporter enzyme The Renilla luciferase assay has
a linear range covering eight orders of magnitude and allows for the detection of approximately 01 femtogram
(approximately 10–21 mole) of Renilla luciferase
1B Format of the DualLuciferase® Reporter Assay
Quantitation of luminescent signal from each of the luciferase reporter enzymes may be performed immediately
following lysate preparation without the need for dividing samples or performing additional treatments The firefly
luciferase reporter assay is initiated by adding an aliquot of lysate to Luciferase Assay Reagent II Quenching of firefly
luciferase luminescence and concomitant activation of Renilla luciferase are accomplished by adding Stop & Glo®
Reagent to the sample tube immediately after quantitation of the firefly luciferase reaction The luminescent signal
from the firefly reaction is quenched by at least a factor of 105 (to ≤0001 residual light output) within 1 second
following the addition of Stop & Glo® Reagent (Figure 4) Complete activation of Renilla luciferase is also achieved
within this 1second period When using a manual luminometer the time required to quantitate both luciferase
reporter activities will be approximately 30 seconds The procedure can be summarized as follows
Elapsed Time
Step 1 Manually add prepared lysate to Luciferase Assay
Reagent II predispensed into luminometer tubes mix
~3 seconds
Step 2 Quantify firefly luciferase activity 12 seconds
Step 3 Add Stop & Glo® Reagent mix 3 seconds
Step 4 Quantitate Renilla luciferase activity 12 seconds
Total elapsed time for the DLR™ Assay 30 seconds
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1B Format of the DualLuciferase® Reporter Assay (continued)
1000000
100000
10000
1000
100
1
010
10
Firefly
Luciferase
Activity
Renilla
Luciferase
Activity
Quenched
Reporter #1
Luminescence
80600
028
116800
Reporter #1 Reporter #2
00004
Residual Activity
Luminescence (RLU)
Figure 4 Measurement of luciferase activities before and after the addition of Stop & Glo® Reagent
The DLR™ Assay allows sequential measurement of firefly luciferase (Reporter #1) followed by Renilla luciferase
activity (Reporter #2) on addition of Stop & Glo® Reagent to the reaction Both reporter activities were quantitated
within the same sample of lysate prepared from CHO cells cotransfected with pGL3 Control Vector (Cat# E1741)
and pRLSV40 Vector (Cat# E2231) To demonstrate the efficient quenching of Reporter #1 by Stop & Glo® Reagent
an equal volume of Stop & Glo® Buffer (which does not contain the substrate for Renilla luciferase) was added Firefly
luciferase luminescence was quenched by greater than 5 orders of magnitude
1C Passive Lysis Buffer
Passive Lysis Buffer (PLB) is specifically formulated to promote rapid lysis of cultured mammalian cells without the
need to scrape adherent cells or perform additional freezethaw cycles (active lysis) Furthermore PLB prevents sample
foaming making it ideally suited for highthroughput applications in which arrays of treated cells are cultured in
multiwell plates processed into lysates and assayed using automated systems Although PLB is formulated for passive
lysis applications its robust lytic performance is of equal benefit when harvesting adherent cells cultured in standard
dishes using active lysis Regardless of the preferred lysis method the release of firefly and Renilla luciferase reporter
enzymes into the cell lysate is both quantitative and reliable for cultured mammalian cells (Figure 5)
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In addition to its lytic properties PLB is designed to provide optimum performance and stability of the firefly and
Renilla luciferase reporter enzymes An important feature of PLB is that unlike other cell lysis reagents it elicits only
minimal coelenterazine autoluminescence Hence PLB is the lytic reagent of choice when processing cells for
quantitation of firefly and Renilla luciferase activities using the DLR™ Assay System Other lysis buffers (eg Glo
Lysis Buffer Cell Culture Lysis Reagent and Reporter Lysis Buffer) either increase background luminescence
substantially or are inadequate for passive lysis If desired the protein content of cell lysates prepared with PLB may
be readily quantitated using a variety of common chemical assay methods Determination of protein content must be
performed using adequate controls Diluting lysates with either water or a buffer that is free of detergents or reducing
agents is recommended in order to reduce the effects that Passive Lysis Buffer may have on background absorbance A
standard curve with BSA must be generated in parallel under the same buffer conditions
Passive Lysis
Active Lysis
120
110
100
90
80
70
60
50
40
30
20
10
0
Firefly Luciferase Activity
Lysis Method
CHO CV1 HeLa NIH3T3
A
Firefly Luciferase Assay
120
110
100
90
80
70
60
50
40
30
20
10
0

Renilla

Luciferase Activity
Lysis Method
CHO CV1 HeLa NIH3T3
B
Renilla Luciferase Assay
1403MA03_6A
Figure 5 Comparison of firefly and Renilla luciferase reporter activities in cell lysates prepared with
Passive Lysis Buffer using either the passive or active lysis procedure Four different mammalian cell types
were cotransfected with firefly and Renilla luciferase expression vectors Lysates were prepared by either exposing
adherent cells to Passive Lysis Buffer for 15 minutes (passive lysis) or scraping adherent cells in the presence of Passive
Lysis Buffer followed by one freezethaw cycle (active lysis) For comparative purposes reporter activities were
normalized to those obtained with the active lysis method for each cell type
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2 Product Components and Storage Conditions
PRODUCT SIZE CAT#
DualLuciferase® Reporter Assay System 100 assays E1910
Each system contains sufficient reagents to perform 100 standard DualLuciferase® Reporter Assays
Includes
• 10ml Luciferase Assay Buffer II
• 1 vial Luciferase Assay Substrate (Lyophilized Product)
• 10ml Stop & Glo® Buffer
• 200µl Stop & Glo® Substrate 50X
• 30ml Passive Lysis Buffer 5X
PRODUCT SIZE CAT#
DualLuciferase® Reporter Assay System 10Pack 1000 assays E1960
Each system contains sufficient reagents to perform 1000 standard DualLuciferase® Reporter Assays using
96well luminometry plates Includes
• 10 × 10ml Luciferase Assay Buffer II
• 10 × 1 vial Luciferase Assay Substrate (Lyophilized Product)
• 10 × 10ml Stop & Glo® Buffer
• 10 × 200µl Stop & Glo® Substrate 50X
• 30ml Passive Lysis Buffer 5X
Note regarding Cat# E1960 For applications requiring more lysis reagent (eg >100µlwell) additional Passive
Lysis Buffer may be purchased separately (Cat# E1941)
Storage Conditions Upon receipt store the DualLuciferase® Reporter Assay System at –20°C Once the Luciferase
Assay Substrate has been reconstituted it should be divided into working aliquots and stored at –20°C for up to
1 month or at –70°C for up to 1 year Ideally Stop & Glo® Reagent (Substrate + Buffer) should be prepared just before
each use If necessary this reagent may be stored at –20°C for 15 days with no decrease in activity If stored at 22°C for
48 hours the reagent’s activity decreases by 8 and if stored at 4°C for 15 days the reagent’s activity decreases by
13 The Stop & Glo® Reagent can be thawed at room temperature up to 6 times with ≤15 decrease in activity
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3 The pGL4 Luciferase Reporter Vectors
3A Description of pGL4 Vectors
The pGL4 Luciferase Reporter Vectors are the next generation of reporter gene vectors optimized for expression in
mammalian cells Numerous configurations of pGL4 Vectors are available including those with the synthetic firefly
luc2 (Photinus pyralis) and Renilla hRluc (Renilla reniformis 5) luciferase genes which have been codon optimized
for more efficient expression in mammalian cells Furthermore both the reporter genes and the vector backbone
including the ampicillin (Ampr) gene and mammalian selectable marker genes for hygromycin (Hygr) neomycin (Neor)
and puromycin (Puror) have been engineered to reduce the number of consensus transcription factor binding sites
reducing background and the risk of anomalous transcription
The pGL4 Vector backbone is provided with either the luc2 or hRluc genes and in certain vectors one or both of two
Rapid Response™ reporter genes The protein levels maintained by these Rapid Response™ luciferase genes respond
more quickly and with greater magnitude to changes in transcriptional activity than their more stable counterparts
For more information on advantages of and improvements made to the pGL4 series of vectors please visit
wwwpromegacompgl4 or see the pGL4 Luciferase Reporters Technical Manual #TM259
3B Important Considerations for CoTransfection Experiments
Firefly and Renilla luciferase vectors may be used together to cotransfect mammalian cells Either firefly or Renilla
luciferase may be used as the control or the experimental reporter gene depending on the experiment and the genetic
contructs available However it is important to realize that trans effects between promoters on cotransfected plasmids
can potentially affect reporter gene expression (6) Primarily this is of concern when either the control or experimental
reporter vector or both contain very strong promoterenhancer elements The occurrence and magnitude of such
effects will depend on the combination and activities of the genetic regulatory elements present on the cotransfected
vectors the relative ratio of experimental vector to control vector introduced into the cells and the cell type
transfected
To help ensure independent genetic expression between experimental and control reporter genes we encourage users
to perform preliminary cotransfection experiments to optimize both the amount of vector DNA and the ratio of
coreporter vectors added to the transfection mix The extreme sensitivity of both firefly and Renilla luciferase assays
and the very large linear range of luminometers (typically 5–6 orders of magnitude) allows accurate measurement of
even vastly different experimental and control luminescence values Therefore it is possible to add relatively small
quantities of a control reporter vector to provide lowlevel constitutive expression of that luciferase control activity
Ratios of 101 to 501 (or greater) for experimental vectorcoreporter vector combinations are feasible and may aid
greatly in suppressing the occurrence of trans effects between promoter elements
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4 Instrument Considerations
4A SingleSample Luminometers
Renilla and firefly luciferases both exhibit stabilized reaction kinetics therefore singlesample luminometers designed
for lowthroughput applications do not require reagent injectors to perform DLR™ Assays Luminometers should be
configured to measure light emission over a defined period as opposed to measuring flash intensity or peak height
For the standard DLR™ Assay we recommend programming luminometers to provide a 2second preread delay
followed by a 10second measurement period However depending on the type of instrument and the number of
samples being processed some users may prefer to shorten the period of premeasurement delay andor the period of
luminescence measurement For convenience it is preferable to equip the luminometer with a computer or an online
printer for direct capture of data output thus eliminating the need to pause between reporter assays to manually
record the measured values Some singletube luminometers equipped with one or two reagent injectors may be
difficult or impossible to reprogram to accommodate the readinjectread format of the DLR™ Assay In such
instances we recommend disabling the injector system and manually adding the reagents
The GloMax® 2020 Luminometers equipped with single or dual autoinjector systems (Cat# E5321 or E5331) are
ideally suited for low to mediumthroughput processing of DLR™ Assays The GloMax® 2020 Luminometer is
preprogrammed to perform injections and to complete sequential readings of both firefly and Renilla luciferase
reporter activities with a single Start command Furthermore the instrument is programmed to provide individual
and normalized luciferase values as well as statistical analyses of values measured within replicate groups
4B MultiSample and PlateReading Luminometers
The most convenient method of performing large numbers of DLR™ Assays is to use a luminometer capable of
processing multiple sample tubes or by configuring assays in a 96well array and using a platereading luminometer
such as the GloMax® 96 Luminometer (Cat# E6511 E6521) For highthroughput applications we recommend first
dispensing the desired volume of each sample into the individual assay tubes or wells of the microplate or preparing
the lysates directly in each well Dualreporter assays are then performed as follows i) inject Luciferase Assay Reagent
II ii) measure firefly luciferase activity iii) inject Stop & Glo® Reagent and iv) measure Renilla luciferase activity
Therefore we recommend multisample and platereading luminometers be equipped with two reagent injectors to
perform the DLR™ Assay Users of highthroughput instruments may be able to perform DLR™ Assays using elapsed
premeasurement and measurement times that are significantly shorter than those prescribed in the standard assay
protocol
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Note Verify that your luminometer provides a diagnostic warning when the luminescence of a given sample exceeds the
linear range of the photomultiplier tube It is common for the luminescence intensity of luciferasemediated reactions to
exceed the linear range of a luminometer If the luminometer does not provide a warning it is important to establish the
luminometer’s linear range of detection prior to performing luciferase reporter assays Purified luciferase (eg Quanti
Lum® Recombinant Luciferase Cat# E1701) or luciferase expressed in cell lysates may be used to determine the working
range of a particular luminometer Perform serial dilutions of the luciferase sample in 1X PLB (refer to Section 5A)
containing 1mgml gelatin The addition of exogenous protein is necessary to ensure stability of the luciferase enzyme at
extremely dilute concentrations
4C Scintillation Counters
In general we do not recommend the use of scintillation counters for quantitating firefly and Renilla luciferase
activities using the integrated DLR™ Assay chemistry Scintillation counters are not equipped with autoinjection
devices therefore samples assayed using the DualLuciferase® format must be processed manually Since the
luminescent signal generated by both luciferases decays slowly over the course of the reaction period (Figure 2) it is
necessary to operate the scintillation counter in manual mode and to initiate each reaction just prior to measurement
This is especially important for the Renilla luciferase reaction which decays more rapidly than the firefly luciferase
reaction As a result of this decay it is also important to control the elapsed time between initiating the reaction and
taking a measurement
If a scintillation counter is used to measure firefly and Renilla luciferase activities configure the instrument so that
all channels are open and the coincidence circuit is turned off This is usually achieved through an option of the
programming menu or by a switch within the instrument If the circuit cannot be turned off a linear relationship
between luciferase concentration and cpm can still be produced by calculating the square root of measured counts per
minute (cpm) minus background cpm (ie [sample – background]12) See Section 6E for a discussion on determining
assay background measurements

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5 Preparation of Cell Lysates Using Passive Lysis Buffer
Two procedures are described for the preparation of cell lysates using PLB The first is recommended for the passive
lysis of cells in multiwell plates The second is intended for those who are harvesting cells grown in culture dishes and
prefer to expedite lysate preparation by scraping the adherent cells In both procedures the firefly and Renilla
luciferases contained in the cell lysates prepared with PLB are stable for at least 6 hours at room temperature (22°C)
and up to 16 hours on ice Freezing of the prepared lysates at –20°C is suitable for shortterm storage (up to 1 month)
however we recommend longterm storage at –70°C Subjecting cell lysates to more than 2–3 freezethaw cycles may
result in gradual loss of luciferase reporter enzyme activities
Materials to Be Supplied by the User
(Solution composition is provided in Section 8A)
• phosphate buffered saline (PBS)
5A Passive Lysis Buffer Preparation
PLB is supplied as a 5X concentrate Prepare a sufficient quantity of the 1X working concentration by adding 1 volume
of 5X Passive Lysis Buffer to 4 volumes of distilled water and mixing well The diluted (1X) PLB may be stored at 4°C
for up to one month however we recommend preparing the volume of PLB required just before use The 5X PLB
should be stored at –20°C
Use only Passive Lysis Buffer for the preparation of cell lysates for use with the DLR™ System PLB is specially
formulated to minimize background autoluminescence
5B Passive Lysis of Cells Cultured in Multiwell Plates
1 Determine transfection parameters (ie plated cell density and subsequent incubation time) such that cells are
no more than 95 confluent at the desired time of lysate preparation Remove the growth medium from the
cultured cells and gently apply a sufficient volume of phosphate buffered saline (PBS) to wash the surface of the
culture vessel Swirl the vessel briefly to remove detached cells and residual growth medium Completely remove
the rinse solution before applying PLB reagent
2 Dispense into each culture well the minimum volume of 1X PLB required to completely cover the cell monolayer
The recommended volumes of PLB to add per well are as follows
Multiwell Plate 1X PLB
6well culture plate 500µl
12well culture plate 250µl
24well culture plate 100µl
48well culture plate 65µl
96well culture plate 20µl

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3 Place the culture plates on a rocking platform or orbital shaker with gentle rockingshaking to ensure complete and
even coverage of the cell monolayer with 1X PLB Rock the culture plates at room temperature for 15 minutes
4 Transfer the lysate to a tube or vial for further handling and storage Alternatively reporter assays may be
performed directly in the wells of the culture plate In general it is unnecessary to clear lysates of residual cell
debris prior to performing the DLR™ Assay However if subsequent protein determinations are to be made
we recommend clearing the lysate samples for 30 seconds by centrifugation at top speed in a refrigerated
microcentrifuge Transfer cleared lysates to a new tube prior to reporter enzyme analyses
Notes
1 Cultures that are overgrown are often more resistant to complete lysis and typically require an increased volume
of PLB andor an extended treatment period to ensure complete passive lysis Firefly and Renilla luciferases are
stable in cell lysates prepared with PLB (7) therefore extending the period of passive lysis treatment will not
compromise reporter activities
2 Microscopic inspection of different cell types treated for passive lysis may reveal seemingly different lysis results
Treatment of many types of cultured cells with PLB produces complete dissolution of their structure within a
15minute period However PLB treatment of some cell types may result in discernible cell silhouettes on the
surface of the culture well or large accumulations of floating debris Despite the appearance of such cell remnants
we typically find complete solubilization of both luciferase reporter enzymes within a 15minute treatment period
(Figure 5) However some types of cultured cells may exhibit greater inherent resistance to lysis and optimizing
the treatment conditions may be required
5C Active Lysis of Cells by Scraping
1 Remove growth medium from the cultured cells and gently apply a sufficient volume of PBS to rinse the bottom
of the culture vessel Swirl the vessel briefly to remove detached cells and residual growth medium Take care to
completely remove the rinse solution before applying the 1X PLB
2 Homogeneous lysates may be rapidly prepared by manually scraping the cells from culture dishes in the presence
of 1X PLB Recommended volumes of PLB to be added per culture dish are listed below
Cell Culture Plate 1X PLB
100 × 20mm culture dish 100ml
60 × 15mm culture dish 400µl
35 × 12mm culture dish 200µl
6well culture plate 250µl
12well culture plate 100µl
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5C Active Lysis of Cells by Scraping (continued)
3 Cells may be harvested immediately following the addition of PLB by scraping vigorously with a disposable
plastic cell lifter or a rubber policeman Tilt the plate and scrape the lysate down to the lower edge Pipet the
accumulated lysate several times to obtain a homogeneous suspension If the scraper is used to prepare more
than one sample thoroughly clean the scraper between uses
4 Transfer the lysate into a tube or vial for further handling and storage Subject the cell lysate to 1 or 2 freezethaw
cycles to accomplish complete lysis of cells Generally it is unnecessary to clear lysates of residual cell debris prior
to performing the DLR™ Assay However if subsequent protein determinations are to be made we recommend
clearing the lysate samples for 30 seconds by centrifugation in a refrigerated microcentrifuge Transfer the cleared
lysates to a fresh tube prior to reporter enzyme analyses
6 DualLuciferase® Reporter Assay Protocol
Materials to Be Supplied by the User
• luminometer
• siliconized polypropylene tube or small glass vial
6A Preparation of Luciferase Assay Reagent II
Prepare Luciferase Assay Reagent II (LAR II) by resuspending the provided lyophilized Luciferase Assay Substrate in
10ml of the supplied Luciferase Assay Buffer II Once the substrates and buffer have been mixed write LAR II on the
existing vial label for easy identification LAR II is stable for one month at –20°C or for one year when stored at –70°C
Do not substitute Luciferase Assay Reagent (Cat# E1483) included in the Luciferase Assay Systems
(Cat# E1500 E1501 E4030 E4530 E4550) for LAR II The Luciferase Assay Reagent supplied with these systems is
not designed for use with the DLR™ Assay System
Notes
1 Repeated freezethawing of this reagent may decrease assay performance We recommend that LAR II be
dispensed into aliquots for each experimental use (eg a 1ml aliquot will provide 10 assays)
2 The components of LAR II are heatlabile Frozen aliquots of this reagent should be thawed in a water bath at
room temperature
3 The process of thawing generates both density and composition gradients within LAR II Mix the thawed reagent
prior to use by inverting the vial several times or by gentle vortexing

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6B Preparation of Stop & Glo® Reagent
Prepare an adequate volume to perform the desired number of DLR™ Assays (100µl reagent per assay) Stop & Glo®
Substrate is supplied at a 50X concentration Add 1 volume of 50X Stop & Glo® Substrate to 50 volumes of Stop & Glo®
Buffer in a glass or siliconized polypropylene tube
Stop & Glo® Reagent (Substrate + Buffer) is best when prepared just before use If stored at 22°C for 48 hours the reagent’s
activity decreases by 8 If necessary Stop & Glo® Reagent may be stored at –20°C for 15 days with no decrease in activity
It may be thawed at room temperature up to 6 times with ≤15 decrease in activity
Note Reagents that have been prepared and stored frozen should be thawed in a room temperature water bath
Always mix the reagent prior to use because thawing generates density and composition gradients
Example 1 (10 assays)
Add 20µl of 50X Stop & Glo® Substrate to 1ml of Stop & Glo® Buffer contained in either a glass vial or siliconized
polypropylene tube This will prepare sufficient Stop & Glo® Reagent for 10 assays
Example 2 (100 assays)
Transfer 10ml Stop & Glo® Buffer into a glass vial or siliconized polypropylene tube Add 200µl of 50X Stop & Glo®
Substrate to the 10ml Stop & Glo® Buffer This will prepare sufficient Stop & Glo® Reagent for 100 DLR™ Assays
6C Standard Protocol
Note The LAR II Stop & Glo® Reagent and samples should be at ambient temperature prior to performing the
DualLuciferase® Assay Prior to beginning this protocol verify that the LAR II and the Stop & Glo® Reagent have
been warmed to room temperature
The assays for firefly luciferase activity and Renilla luciferase activity are performed sequentially using one reaction
tube The following protocol is designed for use with a manual luminometer or a luminometer fitted with one reagent
injector (Figure 6)
Note In some instances it may be desirable to measure only Renilla luciferase reporter activity in the lysates of pGL4
Vectortransfected cells For this application we recommend the Renilla Luciferase Assay System (Cat# E2810 E2820)
If the DLR™ Assay System is used to measure only Renilla luciferase activity it is still necessary to combine 100µl of
both LAR II and Stop & Glo® Reagent with 20µl cell lysate to achieve optimal Renilla luciferase assay conditions

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6C Standard Protocol (continued)
1 Predispense 100µl of LAR II into the appropriate number of luminometer tubes to complete the desired number
of DLR™ Assays
2 Program the luminometer to perform a 2second premeasurement delay followed by a 10second measurement
period for each reporter assay
3 Carefully transfer up to 20µl of cell lysate into the luminometer tube containing LAR II mix by pipetting 2 or
3 times Do not vortex Place the tube in the luminometer and initiate reading
Note We do not recommend vortexing the solution at Step 3 Vortexing may coat the sides of the tube with a
microfilm of luminescent solution which can escape mixing with the subsequently added volume of Stop & Glo®
Reagent This is of particular concern if Stop & Glo® Reagent is delivered into the tube by automatic injection
4 If the luminometer is not connected to a printer or computer record the firefly luciferase activity measurement
5 If available use a reagent injector to dispense 100µl of Stop & Glo® Reagent If using a manual luminometer
remove the sample tube from the luminometer add 100µl of Stop & Glo® Reagent and vortex briefly to mix
Replace the sample in the luminometer and initiate reading
Note It is possible to prime autoinjector systems with little or no loss of DLR™ Assay reagents Before
priming injectors with LAR II or Stop & Glo® assay reagents we recommend first purging all storage liquid
(ie deionized water or ethanol wash solution see Section 6D) from the injector system Priming assay reagent
through an empty injector system prevents dilution and contamination of the primed reagent Thus the volume
of primed reagent may be recovered and returned to the reservoir of bulk reagent
6 If the luminometer is not connected to a printer or computer record the Renilla luciferase activity measurement
7 Discard the reaction tube and proceed to the next DLR™ Assay
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100µl LAR II
100µl Stop & Glo®
Reagent
(inject or vortex)
20µl PLB Lysate
(mix with pipette)
First Measurement
(Firefly Luciferase)
Second Measurement
(Renilla Luciferase)
Figure 6 Format of the DLR™ Assay using a manual luminometer or a luminometer equipped with one
reagent injector If the instrument is equipped with two injectors it may be preferable to predispense the lysate into
luminometer tubes followed by sequential autoinjection of the LAR II and Stop & Glo® Reagents
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6D Important Considerations for Cleaning Reagent Injectors
Proper cleaning of an injector system exposed to Stop & Glo® Reagent is essential if the device is to be later used to
perform firefly luciferase assays by autoinjecting LAR II One of the luciferasequenching components in Stop & Glo®
Reagent has a moderate affinity for plastic materials This compound exhibits a reversible association with the interior
surfaces of plastic tubing and pump bodies commonly used in the construction of autoinjector systems Injector
plumbing that has not been properly cleaned following contact with Stop & Glo® Reagent will leach trace quantities of
quench reagent into solutions that are subsequently passed through the injector system In such cases even very small
quantities of contaminating quench reagent cause significant inhibition of firefly luciferase reporter activity especially
if injectors are used for dispensing more than one type of reagent It is recommended that a particular injector be
dedicated to each of the two reagents and that on completion of a run the wash protocol below be followed to ensure
clean lines Proper cleaning must be performed even when an injector is dedicated for dispensing a single reagent
General Injector Wash Protocol
1 Purge Stop & Glo® Reagent from the injector lines by repeated primingwashing with a volume of deionized
water equivalent to 3 pump void volumes
2 Prepare 70 ethanol as wash reagent Prime the system with at least 5ml of 70 ethanol to completely replace
the void volume and rinse the injector plumbing It is preferable to allow the injector to soak in this wash
solution for 30 minutes prior to rinsing with deionized water
Note The design and materials used in the construction of injector systems varies greatly and some pumps may
require longer than a 30minute soak in the wash reagent to attain complete surface cleaning Luminometers
equipped with Teflon® tubing are not a concern but other tubing such as Tygon® will require an extended soak
time of 12–16 hours (overnight) to ensure complete removal of the Stop & Glo® Reagent from the injector
system
3 Rinse with a volume of deionized water equivalent to at least 3 pump void volumes to thoroughly remove all
traces of ethanol
Wash Protocol for the Injectors in the GloMax® 2020 Luminometer
Trace contamination of Stop & Glo® Reagent may be removed from the GloMax® 2020 Luminometer injector system
as follows
1 Purge Stop & Glo® Reagent from the injector by performing 1 priming cycle with deionized water
2 Perform a flush cycle with 70 ethanol and allow tubing to soak in this wash solution for 30 minutes
3 Perform a flush cycle with deionized water to remove all traces of ethanol

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wwwpromegacom TM040 · Revised 615
6E Determination of Assay Backgrounds
The expression of a luciferase reporter is quantitated as the luminescence produced above background levels In most
cases because the background is exceptionally low luciferase activity is directly proportional to total luminescence
However when measuring very small amounts of luciferase it is important to subtract the background signal from the
measurement of total luminescence The following sections describe how to determine background signals for firefly
and Renilla luciferases respectively
Firefly Luciferase
With rare exceptions all background luminescence in measurements of firefly luciferase arises from the
instrumentation or the sample tubes Background in sample tubes may result from static electricity or from
phosphorescence In particular polystyrene tubes are capable of accumulating significant static buildup that may
contribute to persistent elevated levels of background luminescence Handling and storage of tubes should be done
carefully to minimize static buildup and samples should be handled away from sunlight or very bright lights before
making luminescence measurements
The electronic design of a given luminometer can greatly affect its measurable level of background signal many
luminometers do not read 0 in the absence of a luminescent sample To determine the background signal contributed
by the instrument and sample tube
1 Use Passive Lysis Buffer to prepare a lysate of nontransfected control (NTC) cells
2 Add 100µl of LAR II to 20µl of NTC lysate
3 Measure apparent luminescence activity
The lysates of mammalian cells do not express endogenous luminescence activity the low apparent luminescence in
NTC lysates is the background due to the instrument and possibly the plate or tube holding the luciferase reaction
Be aware that the relative noise in background signals is often quite high Therefore 5–10 readings should be
performed and the mean reading used to obtain a statistically significant value for instrument and plate or tube
background An additional source of high luminescence activity is overflow from an adjacent well when measuring
luminescence in multiwell plates This can be eliminated by using highquality opaque plates that prevent cross talk
Additionally the luminometer mechanics and its ability to read luminescence emitted from individual wells should be
examined before launching an experiment Each instrument differs in its method of injection and luminescence
detection which can play a significant role in cross talk
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TM040 · Revised 615 wwwpromegacom
6E Determination of Assay Backgrounds (continued)
Renilla Luciferase
Background luminescence in the measurement of Renilla luciferase activity can arise from three possible sources
1 Instrument and sample tube or plate background luminescence is similar to that noted above for firefly luciferase
2 Autoluminescence of coelenterazine is caused by nonenzymatic oxidation of the coelenterazine in solution
Although the level of autoluminescence is dependent on solution composition lysates prepared with PLB
generally yield a low and constant luminescence level Stop & Glo® Reagent has been developed with a
proprietary formulation to further reduce autoluminescence Between the effects of the PLB and the Stop & Glo®
Reagent formulations many luminometers are unable to measure the residual autoluminescence
3 Residual luminescence from the firefly luciferase reaction can occur from a small amount of residual
luminescence remaining from the firefly luciferase assay in the DualLuciferase® measurement However since
the firefly luciferase reaction is quenched greater than 100000fold this residual luminescence is only significant
if the Renilla luciferase luminescent reaction is 1000fold less than the intensity of the first firefly luciferase
luminescent reaction
The background luminescence contributed by numbers 1 and 2 above is constant and can be subtracted from all
measurements of Renilla luciferase Because the background from number 3 is variable depending on the expression
of firefly luciferase it may be important to verify that the level of firefly luciferase activity does not yield significant
residual luminescence that may affect the accurate measurement of Renilla luciferase Such a circumstance may arise as
a result of incomplete mixing of the Stop & Glo® Reagent with the sample LAR II mix In addition if the first injection
of LAR II coats the walls of the tube but the second injection with the Stop & Glo® Reagent does not cover the same
exposed surface area inadequate quenching may result
Perform the following steps to determine the background contributed by the instrument sample tube and
coelenterazine autoluminescence
1 Use Passive Lysis Buffer to prepare a lysate of nontransfected control (NTC) cells
2 Add 20µl of the NTC cell lysate to a luminometer tube containing 100µl of LAR II
3 Add 100µl of Stop & Glo® Reagent to the sample tube
4 Measure background
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wwwpromegacom TM040 · Revised 615
Perform the following steps to determine the background from residual firefly luciferase luminescence
1 Use Passive Lysis Buffer to prepare a lysate of cells expressing high levels of firefly luciferase
2 Add 20µl of the cell lysate to a luminometer tube containing 100µl of LAR II
3 Measure firefly luciferase luminescence
4 Add 100µl of Stop & Glo® Reagent
5 Measure apparent luminescence
6 Subtract background contributed from coelenterazine autoluminescence plus instrument background
(as determined above)
For a very strong firefly luciferase reaction the backgroundsubtracted value of quenched luminescence measured
in Step 6 should be 100000fold less than the value of firefly luciferase luminescence measured in Step 3 In most
instances the value for firefly luminescence will not be 100000fold greater than the background value alone
Therefore it is unlikely that significant residual firefly luminescence signal will be detectable above the background
measured in Step 5
7 References
1 Wood KV et al (1984) Synthesis of active firefly luciferase by in vitro translation of RNA obtained from adult
lanterns Biochem Biophys Res Comm 124 592–6
2 de Wet JR et al (1985) Cloning of firefly luciferase cDNA and the expression of active luciferase in Escherichia
coli Proc Natl Acad Sci USA 82 7870–3
3 Wood KV (1991) In Bioluminescence and Chemiluminescence Current Status eds P Stanley and L Kricka
John Wiley and Sons Chichester 11
4 Matthews JC et al (1977) Purification and properties of Renilla reniformis luciferase Biochemistry 16
85–91
5 Lorenz WW et al (1991) Isolation and expression of a cDNA encoding Renilla reniformis luciferase
Proc Natl Acad Sci USA 88 4438–42
6 Farr A and Roman A (1992) A pitfall of using a second plasmid to determine transfection efficiency
Nucleic Acids Res 20 920
7 Sherf BA et al (1996) DualLuciferase® reporter assay An advanced coreporter technology integrating firefly
and Renilla luciferase assays Promega Notes 57 2–9
22 Promega Corporation · 2800 Woods Hollow Road · Madison WI 537115399 USA · Toll Free in USA 8003569526 · 6082744330 · Fax 6082772516
TM040 · Revised 615 wwwpromegacom
8 Appendix
8A Composition of Buffers and Solutions
PBS buffer 10X (per liter)
115g Na2HPO4
2g KH2PO4
80g NaCl
2g KCl
Dissolve in 1 liter of sterile deionized water
The pH of 1X PBS will be 74
8B Related Products
Luminometers
Product Cat#
GloMax® 2020 Luminometer E5311
GloMax® 2020 Luminometer with Single AutoInjector E5321
GloMax® 2020 Luminometer with Dual AutoInjector E5331
GloMax® 96 Microplate Luminometer E6501
GloMax® 96 Microplate Luminometer with Single Injector E6511
GloMax® 96 Microplate Luminometer with Dual Injectors E6521
Luciferase Assay Systems and Reagents
Product Size Cat#
BrightGlo™ Luciferase Assay System 10ml E2610
100ml E2620
10 × 100ml E2650
SteadyGlo® Luciferase Assay System 10ml E2510
100ml E2520
10 × 100ml E2550
DualLuciferase® Reporter 1000 Assay System 1000 assays E1980
Luciferase Assay System 100 assays E1500
1000 assays E1501
Renilla Luciferase Assay System 100 assays E2810
1000 assays E2820
Promega Corporation · 2800 Woods Hollow Road · Madison WI 537115399 USA · Toll Free in USA 8003569526 · 6082744330 · Fax 6082772516 23
wwwpromegacom TM040 · Revised 615
Product Size Cat#
DualGlo® Luciferase Assay System 10ml E2920
100ml E2940
10 × 100ml E2980
EnduRen™ Live Cell Substrate 034mg E6481
34mg E6482
34mg E6485
ViviRen™ Live Cell Substrate 037mg E6491
37mg E6492
37mg E6495
QuantiLum® Recombinant Luciferase 1mg E1701
5mg E1702
Passive Lysis 5X Buffer 30ml E1941
Plasmid DNA Purification Systems
Product Size Cat#
PureYield™ Plasmid Midiprep System 25 preps A2492
100 preps A2495
PureYield™ Plasmid Maxiprep System 10 preps A2392
25 preps A2393
Wizard® Plus SV Minipreps DNA Purification System 50 preps A1330
250 preps A1460
24 Promega Corporation · 2800 Woods Hollow Road · Madison WI 537115399 USA · Toll Free in USA 8003569526 · 6082744330 · Fax 6082772516
TM040 · Revised 615 wwwpromegacom
8B Related Products (continued)
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] Yes luc2A 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
pGL470[hRluc] Yes hRlucB 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 firefly luciferase gene BhRluc synthetic Renilla luciferase gene
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wwwpromegacom TM040 · Revised 615
9 Summary of Changes
The following change was made to the 615 revision of this document
1 The patent information was updated to remove expired statements
(a)US Pat No 5744320 and European Pat No 0 833 939
(b)US Pat Nos 7078181 7108996 and 7118878 European Pat No 1297337 and other patents pending
(c)Certain applications of this product may require licenses from others
© 1996–2015 Promega Corporation All Rights Reserved
DualGlo DualLuciferase GloMax QuantiLum SteadyGlo Stop & Glo and Wizard are registered trademarks of Promega Corporation BrightGlo DLR
EnduRen PureYield Rapid Response and ViviRen are trademarks of Promega Corporation
Teflon is a registered trademark of EI duPont de Nemours and Company Triton is a registered trademark of Union Carbide Chemicals & Plastics
Technology Corporation Tygon is a registered trademark of Norton Performance Plastics Corporation
Products may be covered by pending or issued patents or may have certain limitations Please visit our Web site for more information
All prices and specifications are subject to change without prior notice
Product claims are subject to change Please contact Promega Technical Services or access the Promega online catalog for the most uptodate
information on Promega products

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