Benefits
• Highsensitivity quantitation
of luciferase activity with
6decade linear range
• Automatic data analysis and
calculation of normalized
reporter activity in SoftMax
Pro Software
• SmartInject™ Technology
for reagent mixing with
optimal results
DualLuciferase Reporter (DLR) Assay on
the SpectraMax i3x MultiMode Microplate
Reader with SpectraMax Injector Cartridge
Introduction
Reporter gene assays are used to study
the expression of eukaryotic genes In dual
reporter gene assays cells are transfected
with two vectors the first containing an
experimental reporter gene coupled to
a regulated promoter of interest and
the second containing a control reporter
gene coupled to a constitutive promoter
Normalizing the activity of the experimental
reporter to the control reporter minimizes
experimental variability
Bioluminescent reporter systems using
firefly and Renilla luciferases are widely
used as coreporters because both assays
are easy to run and exquisitely sensitive
Promega’s DualLuciferase® Reporter (DLR)
Assay System allows users to measure
both firefly and Renilla luciferase activity
in a single microplate well with firefly
acting as the experimental reporter and
Renilla the control Figure 1 illustrates the
two enzymatic reactions which take place
sequentially within the same assay well
Firefly luciferase enzyme catalyzes the
oxidation of luciferin with the concomitant
release of light1 The reaction requires
ATP Mg2+ and O2 Renilla luciferase
catalyzes the O2dependent oxidation
of coelenterate luciferin (coelenterazine)
but does not require Mg2+ or ATP2
The enzymes have different substrate
requirements so they can both be
measured in a single reaction mixture
The DLR assay requires delivery of
two separate reagents containing the
different substrates each followed by a
luminescence read This assay workflow
is easily performed using the SpectraMax
i3x MultiMode Microplate Reader with
SpectraMax Injector Cartridge which is
fully DLReady validated3 In this application
note we demonstrate a linear dynamic
APPLICATION NOTE
range of 6 decades for recombinant firefly
and Renilla luciferases as well as linear
detection of luciferasetransfected cells
from 195 to 25000 cells per well
Materials
• DualLuciferase Reporter Assay System
(Promega cat #E1960) contents include
• Luciferase Assay Buffer II
• Luciferase Assay Substrate
• Stop & Glo Buffer
• Stop & Glo Substrate
• 5X Passive Lysis Buffer
• Purified recombinant luciferase enzymes
• Firefly luciferase QuantiLum®
Recombinant Luciferase
(Promega cat #E1701)
• Renilla luciferase Recombinant
Renilla Luciferase (RayBiotech
cat # RB150003P10)
• CHOK1 cells (ATCC cat #CCL61)
Figure 1 Reactions catalyzed by firefly and Renilla luciferases Firefly and Renilla luciferase have
different substrate requirements
luciferin
+ ATP + O2
+ O2
+ AMP + PPi+ CO2 + hv
Mg2+
HO COH firefly
luciferase
N
S N
S
coelenterazine
HO
O OH
N
H
N N
OO
N
S N
S
Renilla
luciferase
HO
O OH
N
N
+ CO2 + hv
+ light
+ light
• Control luciferase vectors
• pGL413[luc2SV40] firefly luciferase
vector (Promega cat #E6681)
• pGL474[hRlucTK] Renilla luciferase
vector (Promega cat #E6921)
• Fugene HD Transfection Reagent
(Promega cat #E2311)
• 6well tissue culture plates
(Corning cat #3516)
• 96well flat clear bottom white TCtreated
microplates (Corning cat #3610)
• BrightMax sealing films
(Genesee cat #12639)
• White 96 and 384well microplates
(Greiner cat #655075 and #781075)
• SpectraMax i3x MultiMode
Microplate Reader
• SpectraMax Injector Cartridge
Methods
Enzyme standard curves
A stock solution of firefly luciferase was
prepared by diluting the 124 mgmL
solution provided to 1 mgmL with 1X
Passive Lysis Buffer (PLB a component
of the DualLuciferase Reporter Assay
System) containing 1 mgmL BSA Stock
Renilla luciferase was prepared by
reconstituting the lyophilized enzyme with
1X PBS to a concentration of 1 mgmL
Working solutions (10 µgmL) of firefly
and Renilla luciferases were made
by transferring 10 µL of stock solution
(1 mgmL) into 990 µL PLB A combined
luciferase stock standard (100 ngmL
each) was then prepared by transferring
10 µL of each luciferase working solution
into 980 µL PLB Serial 110 dilutions of
the combined stock standard yielded
standards with concentrations ranging
from 100 fgmL to 100 ngmL (16 fM to
16 nM) Luciferase Assay Reagent II (LAR
II) and Stop & Glo Reagent were prepared
according to the DualLuciferase Reporter
Assay System technical manual
Injector 1 of the SpectraMax Injector
Cartridge was primed with 260 µL of LAR II
and Injector 2 was primed with 260 µL of
Stop & Glo Reagent In SoftMax Pro the
Acquisition View was used to configure the
plate read with injection (Figure 2) Both
injectors were set to deliver 100 µL (96well
plate) or 25 µL (384well plate) of reagent
using SmartInject Technology which
combines injection with plate shaking for
Figure 2 Acquisition Plan for DLR The Acquisition Plan editor in SoftMax Pro enables a dragand
drop setup of operations to be applied to each well Shown above is the setup for the DLR assay Two
separate inject and read steps were applied SmartInject depicted in the graphic above the sample
Acquisition Plan enables shaking during the injection and 2second delay step so that reagents are
mixed thoroughly and signal develops quickly and consistently from well to well
Dispense and Shake Read
Figure 3 96 and 384well dualluciferase standard curves A 10 dilution series of purified
recombinant firefly (red plot) and Renilla (blue plot) luciferases was assayed using the DLR system A
linear range of 6 decades was measured (r2 0994) Top 96well format bottom 384well format
Luciferase (M)
RLU
Luciferase (M)
RLU
96well plate
384well plate
complete reagent mixing followed by a
2second delay and 10second integration
20 µL (96well plates) or 10 µL (384well
plates) of each luciferase standard was
pipetted into assay wells The assay plate
was placed in the plate drawer of the
SpectraMax i3x reader and the read was
initiated Data analysis and graphing were
performed using SoftMax Pro Software A
preconfigured DualLuciferase Reporter
Assay protocol is included in the SoftMax
Pro protocol library
Cellbased assay
CHOK1 cells were plated at 25x105
cells per well in 6well culture plates and
allowed to attach and grow overnight The
next day cells were transiently transfected
with pGL413[luc2SV40] firefly luciferase
vector and pGL474[hRlucTK] Renilla
luciferase vector following a standard
protocol for the Fugene HD transfection
reagent A ratio of 101 fireflyRenilla vector
DNA was used and each well received
a total of 1 µg DNA and 3 µL Fugene HD
transfection reagent
24 hours after transfection cells were
seeded in a 96well flat clear bottom white
TCtreated microplate at densities from
195 to 25000 cells per well and allowed
to grow overnight They were then lysed
with 1X Passive Lysis Buffer and assayed in
the same plate using the DualLuciferase
Reporter Assay System and SpectraMax
i3x reader with injector cartridge as
described above Prior to assay a solid
white plate seal was applied to the bottom
of the microplate to maximize detection of
luminescence signal
Results
Luciferase standard curves
Signal for firefly and Renilla luciferase
was linear across the 6decade range
of enzyme tested from 16 fM to 16 nM
(Figure 3) For the 96well plate this
equates to 1 fg per well to 1 ng per well
for the 384well plate it is 05 fg per well
to 05 ng per well Both 96 and 384well
assay formats yielded similar linearity and
dynamic range indicating the suitability of
the dualluciferase assay and SpectraMax
i3x system for both formats
Figure 4 Cellbased standard curves Cells transfected with both firefly and Renilla luciferases were
seeded at densities from 195 to 25000 cells per well in a 96well plate and assayed using the DLR
system Results were plotted as RLU vs number of transfected cells seeded per well Red plot firefly
luciferase signal blue plot Renilla luciferase signal (r2 099 for both)
Transfected cells seeded per well
RLU
Figure 5 Firefly luciferase signal in transfected cells was normalized to Renilla luciferase signal
and the resulting normalized values graphed vs number of cells seeded per well
Cells seeded per well
Ratio of fireflyRenilla
Cellbased assay
Linearity of signal for both firefly and Renilla
luciferases was observed across a broad
range of cell densities from 195 to 25000
cells per well (Figure 4) The difference
in magnitude of the luminescence signal
between the two enzymes is due to the
101 ratio of fireflyRenilla vector used to
transfect the cells as well as the different
strengths of the SV40 (firefly vector) and TK
(Renilla vector) promoters
Figure 5 shows the firefly luciferase RLU
values normalized to Renilla luciferase
RLUs The normalized values were similar
across the entire range of cell densities
tested
Contact Us
Phone +18006355577
Web wwwmoleculardevicescom
Email info@moldevcom
Check our website for a current listing
of worldwide distributors
The trademarks used herein are the property of Molecular Devices LLC or their respective owners
Specifications subject to change without notice Patents wwwmoleculardevicescomproductpatents
FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC PROCEDURES
©2015 Molecular Devices LLC
1015
Printed in USA
Conclusion
The results above demonstrate excellent
sensitivity down to < 1 fg per well for firefly
and Renilla luciferases and a 6decade
dynamic range ensuring accurate
measurement of luciferase signal over an
extensive range of luciferase expression
levels and cell densities
The SpectraMax i3x reader features a
cooled photomultiplier tube (PMT) for low
background noise in luminescence When
used in combination with the SpectraMax
Injector Cartridge this system enables
a wealth of flash type luminescence
applications including reporter gene and
other assays with exceptional sensitivity
and dynamic range Analysis is done
rapidly using a preconfigured SoftMax Pro
protocol that displays each luciferase value
and calculates normalized ratios for easy
interpretation of results
References
1 DeLuca MA and WD McElroy (1978) in Meth
Enzymol 533
2 Matthews J C et al (1977) Purification and
properties of Renilla reniformis luciferase
Biochemistry 1658
3 httpswwwpromegacomproductspm
dlreadyluminometersdlreadyvalidated
luminometers
《香当网》用户分享的内容，不代表《香当网》观点或立场，请自行判断内容的真实性和可靠性！
该内容是文档的文本内容，更好的格式请下载文档
• Highsensitivity quantitation
of luciferase activity with
6decade linear range
• Automatic data analysis and
calculation of normalized
reporter activity in SoftMax
Pro Software
• SmartInject™ Technology
for reagent mixing with
optimal results
DualLuciferase Reporter (DLR) Assay on
the SpectraMax i3x MultiMode Microplate
Reader with SpectraMax Injector Cartridge
Introduction
Reporter gene assays are used to study
the expression of eukaryotic genes In dual
reporter gene assays cells are transfected
with two vectors the first containing an
experimental reporter gene coupled to
a regulated promoter of interest and
the second containing a control reporter
gene coupled to a constitutive promoter
Normalizing the activity of the experimental
reporter to the control reporter minimizes
experimental variability
Bioluminescent reporter systems using
firefly and Renilla luciferases are widely
used as coreporters because both assays
are easy to run and exquisitely sensitive
Promega’s DualLuciferase® Reporter (DLR)
Assay System allows users to measure
both firefly and Renilla luciferase activity
in a single microplate well with firefly
acting as the experimental reporter and
Renilla the control Figure 1 illustrates the
two enzymatic reactions which take place
sequentially within the same assay well
Firefly luciferase enzyme catalyzes the
oxidation of luciferin with the concomitant
release of light1 The reaction requires
ATP Mg2+ and O2 Renilla luciferase
catalyzes the O2dependent oxidation
of coelenterate luciferin (coelenterazine)
but does not require Mg2+ or ATP2
The enzymes have different substrate
requirements so they can both be
measured in a single reaction mixture
The DLR assay requires delivery of
two separate reagents containing the
different substrates each followed by a
luminescence read This assay workflow
is easily performed using the SpectraMax
i3x MultiMode Microplate Reader with
SpectraMax Injector Cartridge which is
fully DLReady validated3 In this application
note we demonstrate a linear dynamic
APPLICATION NOTE
range of 6 decades for recombinant firefly
and Renilla luciferases as well as linear
detection of luciferasetransfected cells
from 195 to 25000 cells per well
Materials
• DualLuciferase Reporter Assay System
(Promega cat #E1960) contents include
• Luciferase Assay Buffer II
• Luciferase Assay Substrate
• Stop & Glo Buffer
• Stop & Glo Substrate
• 5X Passive Lysis Buffer
• Purified recombinant luciferase enzymes
• Firefly luciferase QuantiLum®
Recombinant Luciferase
(Promega cat #E1701)
• Renilla luciferase Recombinant
Renilla Luciferase (RayBiotech
cat # RB150003P10)
• CHOK1 cells (ATCC cat #CCL61)
Figure 1 Reactions catalyzed by firefly and Renilla luciferases Firefly and Renilla luciferase have
different substrate requirements
luciferin
+ ATP + O2
+ O2
+ AMP + PPi+ CO2 + hv
Mg2+
HO COH firefly
luciferase
N
S N
S
coelenterazine
HO
O OH
N
H
N N
OO
N
S N
S
Renilla
luciferase
HO
O OH
N
N
+ CO2 + hv
+ light
+ light
• Control luciferase vectors
• pGL413[luc2SV40] firefly luciferase
vector (Promega cat #E6681)
• pGL474[hRlucTK] Renilla luciferase
vector (Promega cat #E6921)
• Fugene HD Transfection Reagent
(Promega cat #E2311)
• 6well tissue culture plates
(Corning cat #3516)
• 96well flat clear bottom white TCtreated
microplates (Corning cat #3610)
• BrightMax sealing films
(Genesee cat #12639)
• White 96 and 384well microplates
(Greiner cat #655075 and #781075)
• SpectraMax i3x MultiMode
Microplate Reader
• SpectraMax Injector Cartridge
Methods
Enzyme standard curves
A stock solution of firefly luciferase was
prepared by diluting the 124 mgmL
solution provided to 1 mgmL with 1X
Passive Lysis Buffer (PLB a component
of the DualLuciferase Reporter Assay
System) containing 1 mgmL BSA Stock
Renilla luciferase was prepared by
reconstituting the lyophilized enzyme with
1X PBS to a concentration of 1 mgmL
Working solutions (10 µgmL) of firefly
and Renilla luciferases were made
by transferring 10 µL of stock solution
(1 mgmL) into 990 µL PLB A combined
luciferase stock standard (100 ngmL
each) was then prepared by transferring
10 µL of each luciferase working solution
into 980 µL PLB Serial 110 dilutions of
the combined stock standard yielded
standards with concentrations ranging
from 100 fgmL to 100 ngmL (16 fM to
16 nM) Luciferase Assay Reagent II (LAR
II) and Stop & Glo Reagent were prepared
according to the DualLuciferase Reporter
Assay System technical manual
Injector 1 of the SpectraMax Injector
Cartridge was primed with 260 µL of LAR II
and Injector 2 was primed with 260 µL of
Stop & Glo Reagent In SoftMax Pro the
Acquisition View was used to configure the
plate read with injection (Figure 2) Both
injectors were set to deliver 100 µL (96well
plate) or 25 µL (384well plate) of reagent
using SmartInject Technology which
combines injection with plate shaking for
Figure 2 Acquisition Plan for DLR The Acquisition Plan editor in SoftMax Pro enables a dragand
drop setup of operations to be applied to each well Shown above is the setup for the DLR assay Two
separate inject and read steps were applied SmartInject depicted in the graphic above the sample
Acquisition Plan enables shaking during the injection and 2second delay step so that reagents are
mixed thoroughly and signal develops quickly and consistently from well to well
Dispense and Shake Read
Figure 3 96 and 384well dualluciferase standard curves A 10 dilution series of purified
recombinant firefly (red plot) and Renilla (blue plot) luciferases was assayed using the DLR system A
linear range of 6 decades was measured (r2 0994) Top 96well format bottom 384well format
Luciferase (M)
RLU
Luciferase (M)
RLU
96well plate
384well plate
complete reagent mixing followed by a
2second delay and 10second integration
20 µL (96well plates) or 10 µL (384well
plates) of each luciferase standard was
pipetted into assay wells The assay plate
was placed in the plate drawer of the
SpectraMax i3x reader and the read was
initiated Data analysis and graphing were
performed using SoftMax Pro Software A
preconfigured DualLuciferase Reporter
Assay protocol is included in the SoftMax
Pro protocol library
Cellbased assay
CHOK1 cells were plated at 25x105
cells per well in 6well culture plates and
allowed to attach and grow overnight The
next day cells were transiently transfected
with pGL413[luc2SV40] firefly luciferase
vector and pGL474[hRlucTK] Renilla
luciferase vector following a standard
protocol for the Fugene HD transfection
reagent A ratio of 101 fireflyRenilla vector
DNA was used and each well received
a total of 1 µg DNA and 3 µL Fugene HD
transfection reagent
24 hours after transfection cells were
seeded in a 96well flat clear bottom white
TCtreated microplate at densities from
195 to 25000 cells per well and allowed
to grow overnight They were then lysed
with 1X Passive Lysis Buffer and assayed in
the same plate using the DualLuciferase
Reporter Assay System and SpectraMax
i3x reader with injector cartridge as
described above Prior to assay a solid
white plate seal was applied to the bottom
of the microplate to maximize detection of
luminescence signal
Results
Luciferase standard curves
Signal for firefly and Renilla luciferase
was linear across the 6decade range
of enzyme tested from 16 fM to 16 nM
(Figure 3) For the 96well plate this
equates to 1 fg per well to 1 ng per well
for the 384well plate it is 05 fg per well
to 05 ng per well Both 96 and 384well
assay formats yielded similar linearity and
dynamic range indicating the suitability of
the dualluciferase assay and SpectraMax
i3x system for both formats
Figure 4 Cellbased standard curves Cells transfected with both firefly and Renilla luciferases were
seeded at densities from 195 to 25000 cells per well in a 96well plate and assayed using the DLR
system Results were plotted as RLU vs number of transfected cells seeded per well Red plot firefly
luciferase signal blue plot Renilla luciferase signal (r2 099 for both)
Transfected cells seeded per well
RLU
Figure 5 Firefly luciferase signal in transfected cells was normalized to Renilla luciferase signal
and the resulting normalized values graphed vs number of cells seeded per well
Cells seeded per well
Ratio of fireflyRenilla
Cellbased assay
Linearity of signal for both firefly and Renilla
luciferases was observed across a broad
range of cell densities from 195 to 25000
cells per well (Figure 4) The difference
in magnitude of the luminescence signal
between the two enzymes is due to the
101 ratio of fireflyRenilla vector used to
transfect the cells as well as the different
strengths of the SV40 (firefly vector) and TK
(Renilla vector) promoters
Figure 5 shows the firefly luciferase RLU
values normalized to Renilla luciferase
RLUs The normalized values were similar
across the entire range of cell densities
tested
Contact Us
Phone +18006355577
Web wwwmoleculardevicescom
Email info@moldevcom
Check our website for a current listing
of worldwide distributors
The trademarks used herein are the property of Molecular Devices LLC or their respective owners
Specifications subject to change without notice Patents wwwmoleculardevicescomproductpatents
FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC PROCEDURES
©2015 Molecular Devices LLC
1015
Printed in USA
Conclusion
The results above demonstrate excellent
sensitivity down to < 1 fg per well for firefly
and Renilla luciferases and a 6decade
dynamic range ensuring accurate
measurement of luciferase signal over an
extensive range of luciferase expression
levels and cell densities
The SpectraMax i3x reader features a
cooled photomultiplier tube (PMT) for low
background noise in luminescence When
used in combination with the SpectraMax
Injector Cartridge this system enables
a wealth of flash type luminescence
applications including reporter gene and
other assays with exceptional sensitivity
and dynamic range Analysis is done
rapidly using a preconfigured SoftMax Pro
protocol that displays each luciferase value
and calculates normalized ratios for easy
interpretation of results
References
1 DeLuca MA and WD McElroy (1978) in Meth
Enzymol 533
2 Matthews J C et al (1977) Purification and
properties of Renilla reniformis luciferase
Biochemistry 1658
3 httpswwwpromegacomproductspm
dlreadyluminometersdlreadyvalidated
luminometers
《香当网》用户分享的内容，不代表《香当网》观点或立场，请自行判断内容的真实性和可靠性！
该内容是文档的文本内容，更好的格式请下载文档