Dual-Glo Luciferase

Technical ManualDual-Glo®Luciferase Assay SystemINSTRUCTIONS FOR USE OF PRODUCTS E2920, E2940 ANDE2980.PRINTED IN USA.Revised 8/09Part# TM058Dual-Glo®Luciferase Assay SystemAll technical literature is available on the Internet at: www.promega.com/tbs Please visit the web site to verify that you are using the most current version of thisTechnical Manual. Please contact Promega Technical Services if you have questions on useof this system. E-mail: techserv@promega.com.1.Description..........................................................................................................12.Product Components and Storage Conditions............................................33.Performing the Dual-Glo®Luciferase Assay...............................................4A.General Considerations.......................................................................................4B.Reagent Preparation.............................................................................................5C.Assay Procedure...................................................................................................64.Related Products................................................................................................75.References...........................................................................................................96.Appendix.............................................................................................................9A.Overview of the Dual-Glo®Luciferase Assay System....................................9B.Development of the Assay................................................................................10Quenching Effect................................................................................................12C.Data Analysis......................................................................................................14Background Subtraction....................................................................................14Normalizing Ratios............................................................................................14Relative Response Ratios ..................................................................................15D.Conditions Affecting Assay Performance......................................................16Culture Medium.................................................................................................18Serum....................................................................................................................19Phenol Red...........................................................................................................20Organic Solvents.................................................................................................20Media Composition............................................................................................22Temperature........................................................................................................261.DescriptionIn cell biology research and pharmaceutical discovery, it is common to test awide variety of experimental conditions or a large number of chemicalcompounds for their effects on cellular physiology (1,2). Traditionally, the easeand sensitivity of firefly luciferase assays have made it relatively simple tomonitor the upregulation of genetic elements. However, it has been moredifficult to measure downregulation of genes because of the difficulty indiscriminating between cell death and cellular downregulation. Normalizingthe expression of an experimental reporter to the expression of a controlreporter can help differentiate between specific and nonspecific cellularresponses. This normalization can also control for transfection efficiencies.Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·www.promega.comPrinted in USA.Revised 8/09Part# TM058Page 11.Description (continued)Firefly and Renillaluciferases are widely used as co-reporters for thesenormalized studies because both assays are quick, easy and sensitive. Fireflyluciferase is a 61kDa and Renillaluciferase a 36kDa protein (3–5). Both aremonomeric and neither requires post-translational processing, so they canfunction as genetic reporters immediately upon translation.The Dual-Glo®Luciferase Assay System(a–e)is designed to allow high-throughput analysis of mammalian cells containing genes for firefly and Renillaluciferases grown in 96- or 384-well plates. The Dual-Glo®Luciferase Reagentcan be added directly to cells in growth medium without washing orpreconditioning. This reagent induces cell lysis and acts as a substrate for fireflyluciferase, producing a stable luminescent signal that can be read over a periodof two hours, with greater than 60% of the luminescent signal retained.Addition of the Dual-Glo®Stop & Glo®Reagent quenches the luminescencefrom the firefly reaction by at least 10,000-fold and provides the substrate forRenillaluciferase in a reaction that can also be read within 2 hours (with asimilar retention in signal). The Dual-Glo®Luciferase Assay System is designedto work in growth media commonly used for mammalian cells with or withoutadded serum. For an overview and information on the development of theDual-Glo®Luciferase Assay System, see Sections 6.A and B.Selected Citations Using the Dual-Glo®Luciferase Assay System:•Perkinton, M.S.et al.(2004) The c-Abl tyrosine kinase phosphorylates the Fe65 adapter protein to stimulate Fe65/amyloid precursor protein nuclear signaling.J. Biol. Chem.279, 22084–91.The authors found that phosphorylation of a phosphotyrosine binding domain,Fe65 by active c-Abl stimulates amyloid precursor protein/Fe65 transcriptionalactivity. GAL4UAS-dependent firefly luciferase and transfection-control Renillaluciferase reporter plasmids were created and propagated in CHO and COS-7cells. The Dual-Glo®Luciferase Assay System was used to monitor firefly andRenillaluciferase activity, with the firefly activity standardized against theRenillaluciferase control activity.•Lin, X. et al.(2005) siRNA-mediated off-target gene silencing triggered by a 7 ntcomplementation. Nucl. Acids Res.33, 4527–35.To identify novel regulators of the HIF-1 pathway the authors performed aknockdown experiment using a synthetic siRNA library against a number ofkinases. Two of their ‘top hits’ directly down-regulated the hif-1αmRNAthrough a 7nt complementation. They built an siRNA library using an HIF-1reporter assay. For siRNA library screening, the HIF-1 reporter and a controlreporter (pRL-TK Vector, Promega) were transfected into H1299 cells. TheDual-Glo®Luciferase Assay System was used to analyze cells transfected withan siRNA against HIF-1αas a positive control and an siRNA irrelevant to theHIF-1 pathway as a negative control.Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·www.promega.comPart# TM058Page 2Printed in USA.Revised 8/09For additional articles that cite the use of the Dual-Glo®Luciferase AssaySystem, visit: www.promega.com/citations/2.Product Components and Storage ConditionsProductSizeCat. #Dual-Glo®Luciferase Assay System10mlE2920Each system contains sufficient components to prepare 10ml of each reagent. Includes:••••10ml1 vial10ml100μlDual-Glo®Luciferase BufferDual-Glo®Luciferase Substrate (lyophilized)Dual-Glo®Stop & Glo®BufferDual-Glo®Stop & Glo®SubstrateProductSizeCat.#®Dual-GloLuciferase Assay System100mlE2940Each system contains sufficient components to prepare 100ml of each reagent. Includes:••••100ml1 vial100ml1,000μlDual-Glo®Luciferase BufferDual-Glo®Luciferase Substrate (lyophilized)Dual-Glo®Stop & Glo®BufferDual-Glo®Stop & Glo®SubstrateProductSizeCat.#®Dual-GloLuciferase Assay System10 × 100mlE2980Each system contains sufficient components to prepare 1,000ml of each reagent.Includes:•10 × 100ml•10 vials•10 × 100ml•10 × 1,000μlDual-Glo®Luciferase BufferDual-Glo®Luciferase Substrate (lyophilized)Dual-Glo®Stop & Glo®BufferDual-Glo®Stop & Glo®SubstrateStorage Conditions:Store the Dual-Glo®Stop & Glo®Substrate and thelyophilized Dual-Glo®Luciferase Substrate at –20°C. The substrates may also bestored at 4°C for up to two weeks. Store the Dual-Glo®Stop & Glo®Buffer andthe Dual-Glo®Luciferase Buffer below 25°C. Buffer storage at roomtemperature is recommended to prevent the need for temperature equilibrationwhen the reagents are reconstituted. Use the reconstituted Dual-Glo®LuciferaseReagent on the day it is prepared or store at –70°C after preparation for up toone month. Prepare the Dual-Glo®Stop & Glo®Reagent on the day it is to beused.Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·www.promega.comPrinted in USA.Revised 8/09Part# TM058Page 32.Product Components and Storage Conditions (continued)!Caution:The lyophilized Dual-Glo®Luciferase Substrate contains dithiothreitol(DTT) and is therefore classified as hazardous. The reconstituted reagent is notknown 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 whenworking with these and all chemical reagents. In addition, the Dual-Glo®Stop & Glo®Substrate contains a highly volatile solvent. Please pipet carefully and close the cap tightly after use.Note:If Dual-Glo®Stop & Glo®Buffer precipitates upon freezing, it can beresolubilized without affecting solution performance. To resolubilize precipitate,store the Dual-Glo®Stop & Glo®Buffer at room temperature for 3 days or at 4°Cfor 2 weeks before use. Precipitate can also be resolubilized by heating to 37°Cfor up to 2 hours with vigorous shaking. Dual-Glo®Stop & Glo®Buffer shouldbe equilibrated to room temperature for use.3.Performing the Dual-Glo® Luciferase AssayThe Dual-Glo®Luciferase Assay System is designed for use in mammalian cellculture medium and is optimized for use with the following types of mediacontaining 0–10% serum: RPMI 1640, DMEM, MEMα, and F12. The reagentshave been developed so that the signals for firefly and Renillaluciferases arerelatively stable. Two hours after addition of the appropriate reagent, the signalof each of the luminescent reactions will generally be at least 60% of their initialsignal intensity at 22°C. Media/sera combinations affect the stability of eachluminescent signal, so experimental determination of assay performance isrecommended for media/sera combinations not listed above (see Section 6.D).The luminescence signal of each reporter also can be affected by the presence ofphenol red, organic solvents or changes in temperature (Section 6.D).Because the luminescent signals are affected by assay conditions, raw resultsshould be compared only between samples measured at the same time andusing the same medium/serum combination. Incorporation of consistentcontrol wells on each plate provides the ability to calculate a normalized fireflyluminescence/Renillaluminescence ratio for each sample well. Thesenormalized ratios will remain essentially constant (±10%) for samples in a platemeasured during the recommended 2-hour measurement window.Incorporating positive and negative control wells within a plate or experimentprovides the ability to calculate a Relative Response Ratio (RRR). The RRR canbe used to compare results between experiments that do not use the samemedia/sera combination or have been affected by changes in temperature orother variables (see Section 6.C).3.A. General ConsiderationsPromega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·www.promega.comPart# TM058Page 4Printed in USA.Revised 8/093.A. General Considerations (continued)Because a small amount of time is required for complete cell lysis and enzymeequilibration, the reagents of the Dual-Glo®Luciferase Assay System should beadded to plates 10 minutes before quantifying luminescence. For maximallight intensity, samples should be measured within 2 hours of reagentaddition. The Dual-Glo®Luciferase Reagents are not designed for use with theautomated injectors that are integrated into some luminometers, as excessivereagent foaming may occur.To achieve linear assay performance at low light levels, the backgroundluminescence must be subtracted from all readings. Some luminometers alsorequire verification of linear response at high light levels (consult yourluminometer instrument manual for usage information).3.B. Reagent Preparation1.Transfer the contents of one bottle of Dual-Glo®Luciferase Buffer to onebottle of Dual-Glo®Luciferase Substrate to create the Dual-Glo®LuciferaseReagent. Mix by inversion until the substrate is thoroughly dissolved.2.Calculate the amount of Dual-Glo®Stop & Glo®Reagent needed to performthe desired experiments. Dilute the Dual-Glo®Stop & Glo®Substrate 1:100into an appropriate volume of Dual-Glo®Stop & Glo®Buffer in a newcontainer.Example:If 6ml of Dual-Glo®Stop & Glo®Reagent is needed, dilute 60μl ofDual-Glo®Stop & Glo®Substrate into 6ml of Dual-Glo®Stop & Glo®Buffer.For Cat.# E2940 and E2980, if the entire bottle of Dual-Glo®Stop & Glo®Bufferis to be used at one time, transfer the entire contents of the Dual-Glo®Stop &Glo®Substrate into the buffer. For Cat.# E2920, combine 10ml Dual-Glo®Stop &Glo®Buffer and 100μl Dual-Glo®Stop & Glo®Substrate.Notes:1.Assay reagents are stable at room temperature for several hours (see Notes2 and 3). Freezing the reagent can reduce the loss of activity of the Dual-Glo®Luciferase Reagent. Do notthaw the reconstituted reagent at temperaturesabove 25°C. Mix well after thawing. The most convenient and effectivemethod for thawing is to place the reagent in a room temperature waterbath. Prepare only the amount of Dual-Glo®Stop & Glo®Reagent required.For best results, prepare the Dual-Glo®Stop & Glo®Reagent immediatelybefore use.2.Approximate stability of Dual-Glo®Luciferase Reagent after reconstitution:6.7% loss in potency after 8 hours at room temperature, 7.8% loss after 24 hours at 4°C, and 10% loss after 1 week at –20°C. This reagent can beexposed to 5 freeze-thaw cycles with little or no negative effect on potency.Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·www.promega.comPrinted in USA.Revised 8/09Part# TM058Page 53.B. Reagent Preparation (continued)3.Approximate stability of Dual-Glo®Stop & Glo®Reagent after reconstitution:8.1% loss after 8 hours at room temperature, 8.5% loss after 24 hours at 4°C.We recommend that the Dual-Glo®Stop & Glo®Reagent always beprepared immediately before use.4.Light intensity is a measure of the rate of catalysis by the luciferases and istherefore temperature sensitive. The temperature optimum for the activityof both luciferases is approximately room temperature (20–25°C), so it isimportant that the reagents be equilibrated to room temperature beforebeginning measurements. To avoid the need to temperature equilibratereagents before use, store the Dual-Glo®Luciferase Buffer and the Dual-Glo®Stop & Glo®Buffer at room temperature. If reagents are colderthan room temperature, place them in a room temperature water bath toequilibrate before use.5.To achieve maximum reproducibility, equilibrate cells in media to roomtemperature before performing luciferase measurements.3.C. Assay Procedure1.Remove multiwell plates containing mammalian cells from the incubator.Make certain that the plates are compatible with the type of luminometerbeing used.2.Measuring firefly luciferase activity:Add a volume of Dual-Glo®Luciferase Reagent equal to the culture medium volume to each well andmix. For 96-well plates, typically 75μl of reagent is added to cells grown in75μl of medium. For 384-well plates, typically 20μl of reagent is added tocells grown in 20μl of medium.3.Wait at least 10 minutes, then measure the firefly luminescence (consultyour luminometer manual for proper use of the instrument). Optimalresults will be generated if the luminescence is measured within 2 hours of addition of Dual-Glo®Luciferase Reagent.4.Measuring Renillaluciferase activity:Add a volume of Dual-Glo®Stop & Glo®Reagent equal to the original culture medium volume to eachwell and mix. As noted in Step 2, this volume is typically 75μl for 96-wellplates and 20μl for 384-well plates.Note:Dual-Glo®Stop & Glo®Reagent should be added to plate wellswithin 4 hours of addition of Dual-Glo®Luciferase Reagent.5.Wait at least 10 minutes, then measure luminescence. Renillaluminescenceshould be measured in the same plate order as the firefly luminescence wasmeasured (Step 3). Optimal results will be generated if the luminescence ismeasured within 2 hours of addition of Dual-Glo®Stop & Glo®Reagent.6.Calculate the ratio of luminescence from the experimental reporter toluminescence from the control reporter. Normalize this ratio to the ratio of Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·www.promega.comPart# TM058Page 6Printed in USA.Revised 8/09a control well or series of control wells that are treated consistently on allplates (see Section 6.C). This normalization provides optimal and consistentresults from the Dual-Glo®Luciferase Assay System. Relative ResponseRatios can then be calculated from the Normalized Ratios. See Section 6.Cfor more information and sample calculations.4.Related ProductspGL4 Luciferase Reporter VectorsPlease visit our Web site (www.promega.com) to see a complete listing of our reportervectors.MultipleProteinReporterMammalianCloningReporterDegradationGeneSelectableVectorRegionGeneSequencePromoterMarkerCat.#pGL4.10[luc2]Yesluc2ANoNoNoE6651pGL4.11[luc2P]Yes“hPESTNoNoE6661pGL4.12[luc2CP]Yes“hCL1-hPESTNoNoE6671pGL4.13[luc2/SV40]No“NoSV40NoE6681pGL4.14[luc2/Hygro]Yes“NoNoHygroE6691pGL4.15[luc2P/Hygro]Yes“hPESTNoHygroE6701pGL4.16[luc2CP/Hygro]Yes“hCL1-hPESTNoHygroE6711pGL4.17[luc2/Neo]Yes“NoNoNeoE6721pGL4.18[luc2P/Neo]Yes“hPESTNoNeoE6731pGL4.19[luc2CP/Neo]Yes“hCL1-hPESTNoNeoE6741pGL4.20[luc2/Puro]Yes“NoNoPuroE6751pGL4.21[luc2P/Puro}Yes“hPESTNoPuroE6761pGL4.22[luc2CP/Puro]Yes“hCL1-hPESTNoPuroE6771pGL4.70[hRluc]YeshRlucBNoNoNoE6881pGL4.71[hRlucP]Yes“hPESTNoNoE6891pGL4.72[hRlucCP]Yes“hCL1-hPESTNoNoE6901pGL4.73[hRluc/SV40]No“NoSV40NoE6911pGL4.74[hRluc/TK]No“NoHSV-TKNoE6921pGL4.75[hRluc/CMV]No“NoCMVNoE6931pGL4.76[hRluc/Hygro]Yes“NoNoHygroE6941pGL4.77[hRlucP/Hygro]Yes“hPESTNoHygroE6951pGL4.78[hRlucCP/Hygro]Yes“hCL1-hPESTNoHygroE6961pGL4.79[hRluc/Neo]Yes“NoNoNeoE6971pGL4.80[hRlucP/Neo]Yes“hPESTNoNeoE6981pGL4.81[hRlucCP/Neo]Yes“hCL1-hPESTNoNeoE6991pGL4.82[hRluc/Puro]Yes“NoNoPuroE7501pGL4.83[hRlucP/Puro]Yes“hPESTNoPuroE7511pGL4.84[hRlucCP/Puro]Yes“hCL1-hPESTNoPuroE7521Aluc2= synthetic firefly luciferase gene. BhRluc= synthetic Renillaluciferase gene.Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·www.promega.comPrinted in USA.Revised 8/09Part# TM058Page 7Luciferase Assay SystemsProductSteady-Glo®Luciferase Assay SystemSize10ml100ml10 × 100ml10ml100ml10 × 100ml100 assays1,000 assays1,000 assays100 assays100 assays1,000 assays1,000 assays1,000 assays1,000 assays100 assays1,000 assays1mg5mg0.34mg3.4mg34mg0.37mg3.7mg37mgCat.#E2510E2520E2550E2610E2620E2650E1910E1960E1980E1500E4030E1501E4530E4550E1483E2810E2820E1701E1702E6481E6482E6485E6491E6492E6495Bright-Glo™ Luciferase Assay SystemDual-Luciferase®Reporter Assay SystemDual-Luciferase®Reporter Assay System 10-PackDual-Luciferase®Reporter 1000 Assay SystemLuciferase Assay SystemLuciferase Assay System with Reporter Lysis BufferLuciferase Assay System 10-PackLuciferase Assay System Freezer PackLuciferase 1,000 Assay SystemLuciferase Assay ReagentRenillaLuciferase Assay SystemQuantiLum®Recombinant LuciferaseEnduRen™ Live Cell SubstrateViviRen™ Live Cell SubstrateLuminometersProductGloMax™ 20/20 Luminometry SystemGloMax™ 20/20 Luminometry System with SingleAuto-InjectorGloMax™ 20/20 Luminometry System with Dual Auto-InjectorGloMax™ 96 Microplate LuminometerGloMax™ 96 Microplate Luminometer with Single InjectorGloMax™ 96 Microplate Luminometer with Dual InjectorSizeeacheacheacheacheacheachCat.#E5311E5321E5331E6501E6511E6521Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·www.promega.comPart# TM058Page 8Printed in USA.Revised 8/09Plasmid DNA Purification SystemProductPureYield™ Plasmid Midiprep SystemPureYield™ Plasmid Maxiprep SystemSize25 preps100 preps10 preps25 prepsCat.#A2492A2495A2392A23935.References1.Alam, J. and Cook, J.L. (1990) Reporter genes: Application to the study of mammaliangene transcription. Anal. Biochem.188, 245–54.2.Wood, K.V. (1991) In: Bioluminescence and Chemiluminescence: Current Status, Stanley,P., and Kricka, L., eds., John Wiley and Sons, Chichester, NY, 543.3.Wood, K.V. et al.(1984) Synthesis of active firefly luciferase by in vitro translation ofRNA obtained from adult lanterns. Biochem. Biophys. Res. Comm.124, 592–6.4.deWet, J.R. et al.(1985) Cloning of firefly luciferase cDNA and the expression ofactive luciferase in Escherichia coli. Proc. Natl. Acad. Sci.USA82, 7870–3.5.Matthews. J.C. et al.(1977) Purification and properties of Renilla reniformisluciferase.Biochemistry16, 85–91.6.AppendixTranscriptional regulation coupled to reporter gene expression is routinelyused to study a wide range of physiological responses. A common example isthe analysis of receptor function by quantitating the action of specific reporterresponse elements on gene expression. Firefly luciferase has been used as asimple, convenient and sensitive reporter gene for this type of analysis.An increase in the transcription or translation of a reporter molecule, likefirefly luciferase, is easily tracked in a biological system. Firefly luciferase isimmediately functional upon translation. Thus if the amount of luminescencefrom an experimental sample is greater than the luminescence from a controlsample, an increase in transcription or translation has occurred.Studies of antagonists or other factors that decrease transcription or translationare difficult to interpret when using a single reporter gene. A decrease in anexperimental reporter response (firefly luciferase in this case) can be caused bya specific effect on the reporter or by a global effect such as cell death.Normalization of an experimental reporter with a control reporter from thesame sample, such as Renillaluciferase, allows a distinction to be madebetween specific and global effects. A decrease in firefly luminescence withRenillaluminescence remaining unchanged indicates a specific impact of theexperimental condition. A decrease of both luminescences indicates a globalimpact on the cell or cell population (i.e., cell death, inhibition of cell growth,variable initial cell numbers).6.A. Overview of the Dual-Glo® Luciferase Assay SystemPromega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·www.promega.comPrinted in USA.Revised 8/09Part# TM058Page 96.A. Overview of the Dual-Glo® Luciferase Assay System (continued)The Dual-Luciferase®Reporter Assay System (DLR™ Assay System, Cat.# E1910) allows measurement of both firefly and Renillaluciferases from asingle sample. This assay system, however, generates luminescence that rapidlydecreases in intensity. The firefly luciferase signal decreases 50% inapproximately 12–15 minutes, and the Renillaluciferase signal decreases 50% inless than 3 minutes. These signal kinetics make the measurement of firefly andRenillaluciferases difficult if large numbers of samples are to be measured in96- or 384-well plates. Although robotic systems can easily add reagents to allthe samples on a plate at the same time, the signals are too unstable to easilymeasure the luminescence from the entire plate without having to calculatetime corrections.The Dual-Glo®Luciferase Assay System, with its stabilized luminescent signals(each signal retains at least 60% of its initial luminescence after 2 hours), caneasily be used to measure both firefly and Renillaluminescence in multiwellplates. Many plates containing experimental samples can be batch processed;all plates can be filled with reagent at one time, and the luminescence fromeach plate can be measured in series. In this way, the Dual-Glo®Luciferase Assay System can easily and rapidly quantitate bothfirefly and Renillaluciferases, allowing one to distinguish between global andspecific effects.6.B. Development of the AssayFirefly and Renillaluciferases have distinct evolutionary origins and have verydifferent enzyme structures and substrates. Promega has been able to exploitthese differences so that the two luciferases can be measured in succession,with firefly luciferase luminescence elicited by one reagent, while a secondreagent simultaneously quenches the firefly luciferase and elicits Renillaluciferase luminescence.Firefly and Renillaluciferases do not require post-translational modification andthus are fully functional enzymes immediately after translation (3,4). In orderto generate luminescence firefly luciferase requires beetle luciferin, ATP,magnesium and molecular oxygen. Renillaluciferase requires only coelenterateluciferin (coelenterazine) and molecular oxygen (Figure 1). Firefly and Renillaluciferases undergo spontaneous inactivation after generating luminescence.This inactivation causes the “flash”-type kinetics seen in assays that have beenoptimized for maximal assay sensitivity, like the DLR™ Assay System (Figure 2).In order to generate luminescence with signal stability that is amenable torobotic measurements, the rate of inactivation and subsequently the rate ofcatalysis must be slowed. For this reason, “glo”-type reagents developed foruse in high-throughput screening, like the Steady-Glo®and Dual-Glo®Reagents, have lower luminescences than “flash”-type reagents that have been developed for maximal sensitivity, like the Luciferase and DLR™ AssayReagents.Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·www.promega.comPart# TM058Page 10Printed in USA.Revised 8/09HOSNNSCOOH+ATP+O2Recombinant FireflyLuciferaseMg2+OSNNSO+AMP+PPi+CO2+LightBeetle LuciferinOxyluciferinONNHHONOH+O2RenillaLuciferaseOHONNHNHO1399MA03_6A+CO2+LightCoelenterazineCoelenteramideFigure 1. Bioluminescent reactions catalyzed by firefly and Renillaluciferases.Mono-oxygenation of beetle luciferin is catalyzed by firefly luciferase in thepresence of Mg2+, ATP and molecular oxygen. Unlike beetle luciferin, coelenterazineundergoes mono-oxygenation catalyzed by Renillaluciferase but requires onlymolecular oxygen.A.Firefly Luminescence (RLU)10,000B.Renilla Luminescence (RLU)10,000DLR™Dual-Glo™1,0001,000100100020406080100120020406080100120Time (Minutes)Time (Minutes)Figure 2. Comparison of the signal decay of luciferases in Dual-Glo® Reagent and DLR™ Assay Reagent.The two luciferase assay reagents were compared using1.67 × 10–9M (plus 1mg/ml gelatin) firefly and Renillaluciferase (20μl and 100μl ofenzyme mix were used with DLR™ and Dual-Glo®Reagents, respectively). Theluciferases were diluted in RPMI 1640 before assaying with Dual-Glo®LuciferaseReagent and in Passive Lysis Buffer (Cat.# E1941) before assaying with DLR™Assay Reagent. The DLR™ Assay measurements were taken immediately afterreagent addition, while the Dual-Glo®measurements were taken after a 10-minuteincubation at room temperature. Luminescence was integrated over 0.5 seconds per well at regular intervals until 2 hours after reagent addition. Panel A.Fireflyluciferase activity in Dual-Glo®and DLR™ Assays. Panel B.Renillaluciferaseactivity in Dual-Glo®and DLR™ Assays.Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·www.promega.comPrinted in USA.Revised 8/09Part# TM058Page 113619MA02_2A10106.B. Development of the Assay (continued)Quenching EffectProviding the ability to measure firefly luminescence is only one of therequirements for the Dual-Glo®Luciferase Assay System. After measurementof the firefly luminescence, the Dual-Glo®Stop & Glo®Reagent mustsimultaneously quench firefly luminescence and generate Renillaluminescence.The Dual-Glo®Stop & Glo®Reagent has been designed to decrease theluminescence of the firefly luciferase reaction by at least 10,000-fold (Figure 3).Thus if the firefly luciferase luminescence generated 530,000 Relative LightUnits per second, the maximum firefly luminescence measured 10 minutesafter the Dual-Glo®Stop & Glo®Reagent addition would be 53 Relative LightUnits per second. The promoter for the control reporter (either firefly or Renillaluciferase) should be chosen so that the luminescence from the firefly luciferaseis no more than 100 times the luminescence from the Renillaluciferase. If this isthe case, the firefly luciferase luminescence will contribute no more than 1% tothe Renillaluciferase luminescence, and variability in the firefly luciferasesignal will not noticeably affect the Renillaluciferase signal.10,000,0001,900,000170,000Luminescence (RLU)1,000,000100,00010,0001,00010010(60,000-foldquench)303620MA02_2AFirefly LuciferaseActivityQuenchedLuminescenceRenillaLuciferaseActivityFigure 3. Measurement of luciferase activities before and after addition of Dual-Glo® Stop & Glo®Reagent.Both firefly and Renillaluciferase reporteractivities were quantitated using a 100μl sample of purified firefly or Renillaluciferase (1.67 × 10–9M and 1.67 × 10–10M, respectively, with 1mg/ml gelatin).The concentration of Renillaluciferase is 10-fold less than that of fireflyluciferase. To demonstrate the efficient quenching of firefly luciferase by Dual-Glo®Stop & Glo®Reagent, an equal volume of Dual-Glo®Stop & Glo®Buffer(which does not contain the substrate for Renillaluciferase) was added to theDual-Glo®Luciferase Reagent with firefly luciferase. Firefly luciferaseluminescence was quenched by greater than 4 orders of magnitude with0.0016% residual activity. RLU = relative light units.Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·www.promega.comPart# TM058Page 12Printed in USA.Revised 8/09The Dual-Glo®Stop & Glo®Reagent has been designed to provide simul-taneous quenching of the firefly luciferase signal and sustained luminescencefor the Renillaluciferase reaction. Renillaluciferase does not use any of thesubstrates for the firefly reaction except molecular oxygen. This permits theDual-Glo®Stop & Glo®Reagent to both quench the firefly reaction andgenerate the Renillaluciferase reaction. Renillaluciferase is similar to fireflyluciferase in that the enzyme inactivates itself. As with the firefly luciferase,the rate of catalysis must be slowed if the luminescent signal is to be stabilized.“Glo”-type Renillaassays (Dual-Glo®Luciferase Assay) are therefore not assensitive as “flash”-type assays (DLR™ and RenillaLuciferase Assays).Unlike the firefly luciferase substrate (beetle luciferin), coelenterazine willauto-oxidize in the absence of Renillaluciferase. This enzyme-independentreaction is termed autoluminescence. Reaction conditions such as highdetergent levels created in homogeneous assays can increase the level ofautoluminescence, which can limit the sensitivity of the Renillaluciferasereporter system. The Dual-Glo®Luciferase Assay System uses uniquetechnology that reduces autoluminescence to levels that are often undetectable,thereby increasing the sensitivity of the Renillaluciferase reagent of the Dual-Glo®Luciferase Assay System by >100-fold over nonoptimized reagents(Figure 4).Optimized ReagentNonoptimized Reagent1,000,000100,00010,0001,000100101Renilla LuciferaseLuminescence (RLU)Nonoptimized AutoluminescenceOptimized Autoluminescence3621MA02_2A0.110–1910–1810–1710–1610–1510–1410–1310–12[Renilla Luciferase] (M)Figure 4. The Dual-Glo® Luciferase Assay System is optimized for minimalautoluminescence from Renillaluciferase substrate.Luminescence from a titrationof Renillaluciferase was compared in Dual-Glo®Reagent before and afteroptimization for homogeneous assay formats. Luciferase concentrations varied overa titration range of 1 × 10–12to 1 × 10–19moles/reaction. One hundred microliters ofRenillaluciferase in RPMI 1640 (containing 1mg/ml gelatin) was added to 100μl ofDual-Glo®Luciferase Reagent and 100μl of Dual-Glo®Stop & Glo®Reagent, eitheroptimized or nonoptimized for homogeneous assay format. Samples were incubatedfor 10 minutes at 22°C, then measured on a Turner Designs Model 20e luminometer.Light emission was integrated over 5 seconds after an initial 2-second preread delay.Limit of detection values shown (horizontal lines labeled Optimized andNonoptimized) represent background plus three standard deviations and weredetermined for each assay by performing the assay without enzyme.Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·www.promega.comPrinted in USA.Revised 8/09Part# TM058Page 136.C. Data AnalysisBackground SubtractionFor maximal accuracy, the luminescence measurements of both firefly andRenillaluciferases should be background-subtracted. Neither enzyme isendogenously expressed in mammalian cells, so the source of backgroundluminescence is either a characteristic of the luminometer (backgroundmeasurements can be as low as 0.000–0.001RLU or as high as 300RLU) or of theluminescent substrate. Beetle luciferin, one of the firefly luciferase substrates,does not generate light in the absence of luciferase in the Dual-Glo®Reagents. Aproprietary chemistry minimizes the enzyme-independent luminescence(autoluminescence) exhibited by coelenterate luciferin, the substrate for Renillaluciferase in the Dual-Glo®Stop & Glo®Reagent. Background luminescence forboth luciferase reagents therefore may not be measurable above thebackground on many luminometers.Background measurements for firefly luciferase should be taken from samplesconsisting of nontransfected cells and Dual-Glo®Luciferase Reagent. For Renillaluciferase, background measurements should be taken from samples containingnontransfected cells and both Dual-Glo®Luciferase Reagent and Dual-Glo®Stop & Glo®Reagent. Sample volumes for background measurements must bethe same as experimental sample volumes and contain the same media/sera combinations as the experimental samples.Normalizing RatiosNormalizing the results from each experimental sample to control samplesrepeated on each plate minimizes the impact of variables like temperature,plate order and timing on the ratio of experimental/control reporter activity.Example 1:An experiment includes 20 plates. If the timing of measurement of the plates ischanged between the firefly and Renillaluminescence measurements, then theratio of firefly luminescence to Renillaluminescence will be different on each ofthe plates because the two reporter signals would have decayed by differentamounts (changing the order of the plates changes the length of time betweenreagent addition and plate reading). For instance, the luminometer jammed 20minutes after the Dual-Glo®Stop & Glo®Reagent was added to a large stack ofplates:Ratio of plate 1 control firefly luminescence/Renillaluminescence = 1.7.Ratio of plate 1 well A1 firefly luminescence/Renillaluminescence = 3.4.Both firefly luciferase and Renillaluciferase were measured 10 minutes after thereagents were added.Relative ratios are: control = 1.7/1.7 = 1 and well A1 = 3.4/1.7 = 2.However, for plate 12 the firefly luciferase was measured 40 minutes afterDual-Glo®Luciferase Reagent was added, but because of the luminometer jam,Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·www.promega.comPart# TM058Page 14Printed in USA.Revised 8/09the Renillaluciferase was not measured until 75 minutes after the Dual-Glo®Stop& Glo®Reagent was added.Ratio of plate 12 control firefly luminescence/Renillaluminescence = 2.1.Ratio of plate 12 well A1 firefly luminescence/Renillaluminescence = 4.2.Relative ratios are: control = 2.1/2.1 = 1 and well A1 = 4.2/2.1 = 2.Example 2:The Dual-Glo®Assay is being performed in a cell culture medium (MEMα)that causes the firefly and Renillaluciferases to decay at different rates (fireflyluciferase retains ~90% activity over 2 hours, Renillaluciferase retains ~75%activity over 2 hours). Normalizing the ratios permits comparison of theseratios between plates in the same experiment.Ratio of plate 1 control firefly luminescence/Renillaluminescence = 6.4.Ratio of plate 1 well A1 firefly luminescence/Renillaluminescence = 1.6.Both firefly luciferase and Renillaluciferase were measured 10 minutes afterthe reagents were added.Relative ratios were: control = 6.4/6.4 = 1 and well A1= 1.6/6.4 = 0.25.For plate 12, both firefly luciferase and Renillaluciferase were measured 65minutes 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 firefly luminescence/Renillaluminescence = 6.9.Ratio of plate 12 well A1 firefly luminescence/Renillaluminescence = 1.7.Relative ratios are: control = 6.9/6.9 = 1 and well A1 = 1.7/6.9 = 0.25.Other factors may affect the ratio of experimental/control reporter. As seen in Section 6.D and mentioned above, chemical components of themedium/serum combination affect the firefly luciferase signal stabilitydifferently than they affect the Renillaluciferase signal stability. This is alsotrue of changes in temperature and other parameters that affect enzymereactions. Normalization of the experimental/control reporter activity isessential for easy comparison of samples with minimal variability across anexperiment.Relative Response RatiosA Relative Response Ratio (RRR) can be determined to assist in quantitation ofthe impact of an experimental treatment on reporter gene expression. The RRRpermits the comparison of multiple treatments from different experimentsbecause it provides a framework within which the effect of the treatment canbe placed.Calculation of RRR requires the inclusion of 2 sets of controls on each plate: apositive control that provides maximal luminescence, and a negative controlthat provides minimal luminescence. For an experiment that monitorsdownregulation of the experimental reporter by chemical treatments, the Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·www.promega.comPrinted in USA.Revised 8/09Part# TM058Page 156.C. Data Analysis (continued)positive control for the experimental series might be no treatment. The negativecontrol would be treatment with some compound that had previously beenshown to drastically inhibit the experimental reporter. If these 2 samples areincluded on every plate, the impact of each new compound can bequantitatively evaluated by its influence on the experimental reporter within the context of the positive and negative control.Example:If the ratio of experimental reporter luminescence/control reporterluminescence is 53 for the positive control, 1.3 for the negative control, and 22for the experimental treatment, all of these values can be scaled so that thepositive control is assigned the value of 1 and the negative control is assignedthe value of 0. The RRR for each experimental treatment can then be calculatedusing this formula:(experimental sample ratio) – (negative control ratio)RRR = (positive control ratio) – (negative control ratio)The RRR for the experimental treatment example listed above would be:22 – 1.3RRR = = 0.40 or 40%53 – 1.3The experimental compound is 40% as effective as the negative control atdecreasing expression of the experimental reporter at this concentration. Ahierarchy of efficacy for the experimental system would be: i) no treatmentgenerated a RRR of 100%; ii) the experimental compound generated a RRR of40% and; iii) the negative control generated a RRR of 0%. Those compoundsthat are most significant will have RRRs that are negative, since they would bemore effective inhibitors than the negative control.Note:If the absolute luminescence values are close to background, theluminescences will need to be background subtracted before the ratios arecalculated.6.D. Conditions Affecting Assay PerformanceThe data presented in this section are intended to provide a general overviewof assay characteristics under a wide range of experimental conditions. The cellculture medium accounts for 50% of the firefly luciferase and 33% of the Renillaluciferase reaction volumes. Thus medium can affect assay performance. Thechemistry used for the Dual-Glo®Luciferase Assay System is unique whencompared to other assays developed for high-throughput analysis andcompared to the DLR™ Assay System. Therefore the data presented in thistechnical manual may not be applicable to other luciferase assay systems.Purified firefly and Renillaluciferases were diluted into culture medium togenerate the data presented in this section. This was done to illustrate theperformance characteristics without adding the experimental complexitiesPromega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·www.promega.comPart# TM058Page 16Printed in USA.Revised 8/09common to cell culture. However, as shown in Figure 5, purified luciferasesdiluted into culture medium show little or no difference when compared toenzymes expressed in transfected cells. Gelatin (1mg/ml) was added to thewells to simulate the protein that would normally be contributed by cells.Addition of gelatin is not required when using the Dual-Glo®Luciferase AssaySystem with cells.A.Relative Firefly Luminescence (Percent Remaining After 10 Minutes)100806040200B.Relative Firefly Luminescence (Percent Remaining After 10 Minutes)100806040200CHO LysatePurified Luciferase293 LysatePurified Luciferase020406080100120020406080100120Time (Minutes)C.Relative Renilla Luminescence (Percent Remaining After 10 Minutes)100806040200Time (Minutes)D.Relative Renilla Luminescence (Percent Remaining After 10 Minutes)100806040200CHO LysatePurified Luciferase293 LysatePurified Luciferase020406080100120020406080100120Time (Minutes)Time (Minutes)Figure 5. Reaction kinetics for purified firefly and Renillaluciferase and forluciferases expressed by transiently transfected mammalian cells.Samples in 96-well plates consisted of 75μl of either purified firefly and Renillaluciferases (bothat 1.67 × 10–9M, 1mg/ml gelatin as a protein carrier) or mammalian cells transfectedwith firefly and Renillaluciferase genes (using pCI Mammalian Expression Vector[Cat.# E1731] with luc+ inserted at XbaI/XhoI sites for firefly luciferase and thephRL-SV40 Vector for Renillaluciferase [Cat.# E6261]) in the same growth medium.Seventy-five microliters of Dual-Glo®Luciferase Reagent was added to plate wellsand mixed on a plate shaker. After a 10-minute room temperature incubation,luminescence was integrated over 0.5 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 luciferasewas measured in CHO cells in F12 medium (Panel A) and in 293 cells in DMEM(Panel B). Renillaluciferase 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 inrelative luminescence is apparent over time between transfected cells and purifiedenzyme in the same culture medium. Number of samples = 6 for cell lysate data, 3for purified enzyme data.Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·www.promega.comPrinted in USA.Revised 8/09Part# TM058Page 173622MA02_2A6.D. Conditions Affecting Assay Performance (continued)Culture MediumWhen performing the Dual-Glo®Luciferase Assay, the culture medium andany compounds added to the medium make up half of the chemicalenvironment of the firefly luciferase reaction, and one-third of the environmentof the Renillaluciferase reaction. Although the Dual-Glo®Reagents aredesigned to work with many common culture media, compositional differencesbetween the different media may affect the assay characteristics (i.e., lightintensity and signal stability).The Dual-Glo®Luciferase Reagents were designed to provide relatively highluminescence with signals that retain at least 60% of their initial luminescenceafter 2 hours when used with common growth medium. However,performance differences are evident between these media (Figure 6), as well as between the same media from different manufacturers. Although these differences are generally small and do not diminish the utility of theDual-Glo®Luciferase Assay System, controls should be incorporated into everybatch of plates to correct for this variability.A.B.Signal Stability (Percent Luminescence Remaining After 120 Minutes)FireflyRenilla1008060402003623MA02_2ARelative Luminescence (Percent)100806040200DMEMF12MEMαRPMIDMEMF12MEMαRPMIFigure 6. Relative luminescence intensity and signal stability of firefly and Renillaluciferase in four common media.Purified firefly or Renillaenzyme (1.67 × 10–9M, 1mg/ml gelatin) was added to a 96-well plate at 100μl per well.Dilutions were made in RPMI 1640, DMEM, MEMαor F12 medium. Either Dual-Glo®Luciferase Reagent (for firefly luciferase) or Dual-Glo®Luciferase Reagentplus Dual-Glo®Stop & Glo®Reagent (for Renillaluciferase) was added, andluminescence measurements were integrated over 0.5 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 bothfirefly and Renillaluciferases. Panel B.Signal stability in various media expressed aspercent luminescence remaining after 2 hours. Number of samples = 3; relativestandard error ≤3.4%.Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·www.promega.comPart# TM058Page 18Printed in USA.Revised 8/09SerumThe Dual-Glo®Reagents are compatible with medium containing serum. Thereagents have been designed for use with serum concentrations from 0–10%,and the luminescent signals generated are minimally affected by the presenceof fetal bovine or calf serum (Figure 7).Relative Luminescence (Percent)1201008060402000246810Signal Stability (Percent Luminescence Remaining After 120 Minutes)12010080604020002468CSFBS10[Serum] (Percent of Medium)[Serum] (Percent of Medium)C.Relative Luminescence (Percent)1201008060402000246810D.Signal Stability (Percent Luminescence Remaining After 120 Minutes)1201008060402002468CSFBS10[Serum] (Percent of Medium)[Serum] (Percent of Medium)Figure 7. Effects of serum on luminescence intensity and signal stability.Purifiedfirefly or Renillaluciferase (1.67 × 10–9M, 1mg/ml gelatin) was added to a 96-wellplate at 100μl per well. F12 medium containing various concentrations of either fetalbovine serum (FBS) or calf serum (CS) was used for the assay. Either Dual-Glo®Luciferase Reagent (for firefly luciferase) or Dual-Glo®Luciferase Reagent plusDual-Glo®Stop & Glo®Reagent (for Renillaluciferase) was added, and luminescencemeasurements were integrated over 0.5 seconds per well. Firefly luciferase relativeluminescence (Panel A) and signal stability (Panel B) were measured in mediumcontaining fetal bovine serum (FBS) and calf serum (CS). Renillaluciferase relativeluminescence (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 samplescontaining no serum. Signal stability expressed as percent of luminescence remaining after 2 hours. Number of samples = 3; relative standard error ≤3.9%.Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·www.promega.comPrinted in USA.Revised 8/09Part# TM058Page 193624MA02_2A06.D. Conditions Affecting Assay Performance (continued)Phenol RedPhenol red is a pH indicator commonly used in cell culture media. Manycommercial media formulations contain 5–15mg/L phenol red, causing thecharacteristic red color. This compound can reduce assay sensitivity (Figure 8).However, in most applications the presence of phenol red will not significantlyaffect the utility of the Dual-Glo®Luciferase Assay System. To minimize itseffect, use as little phenol red as possible in culture medium.A.B.Firefly LuciferaseRenilla Luciferase10080604020010203625MA02_2A10080604020001020Signal Stability (Percent LuminescenceRemaining After 120 Minutes)Relative Luminescence (Percent)0[Phenol Red] (mg/L)[Phenol Red] (mg/L)Figure 8. The effect of phenol red on luminescence intensity and signal stability.Purified firefly or Renillaluciferase (1.67 × 10–9M, 1mg/ml gelatin) was added to a96-well plate at 100μl per well. MEMαcontaining various concentrations of phenolred was used for the assay. Either Dual-Glo®Luciferase Reagent (for fireflyluciferase) or Dual-Glo®Luciferase Reagent plus Dual-Glo®Stop & Glo®Reagent (forRenillaluciferase) was added, and luminescence measurements were integrated over0.5 seconds per well. Luminescence is compared for samples containing phenol redand for samples containing no phenol red. Panel A.Luminescence is shown relativeto that measured without phenol red. Panel B.Signal stability in variousconcentrations of phenol red expressed as percent of luminescence remaining after 2 hours. Number of samples = 3; relative standard error ≤4.0%.Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·www.promega.comPart# TM058Page 20Printed in USA.Revised 8/09Organic SolventsOrganic solvents are typically present in reporter gene assays, because they areused to stabilize and solubilize screening compounds. DMSO, ethanol andmethanol have little effect on the assay (Figure 9). The compatibility of othersolvents should be verified prior to use.Relative Luminescence (Percent)1201008060402000123Signal Stability (Percent LuminescenceRemaining After 120 Minutes)12010080604020001DMSOMethanolEthanol23[Solvent] (Percent of Media)[Solvent] (Percent of Media)C.D.Relative Luminescence (Percent)1201008060402000123Signal Stability (Percent LuminescenceRemaining After 120 Minutes)12010080604020001DMSOMethanolEthanol3626MA02_2A23[Solvent] (Percent of Media)[Solvent] (Percent of Media)Figure 9. The effect of organic solvents on luminescence intensity and signalstability.Purified firefly or Renillaluciferase (1.67 × 10–9M, 1mg/ml gelatin)was added to a 96-well plate at 100μl per well. F12 medium containing variousconcentrations of DMSO, methanol or ethanol was used for the assay. Either Dual-Glo®Luciferase Reagent (for firefly luciferase) or Dual-Glo®LuciferaseReagent plus Dual-Glo®Stop & Glo®Reagent (for Renillaluciferase) wasadded, and luminescence measurements were integrated over 0.5 seconds perwell. Firefly luciferase relative luminescence (Panel A) and signal stability(Panel B), as well as Renillaluciferase relative luminescence (Panel C) andsignal stability (Panel D) were determined in various concentrations ofsolvent. Luminescence is expressed relative to samples without organicsolvents. Signal stability is expressed as percent of luminescence remainingafter 2 hours. Number of samples = 3; relative standard error ≤4.0%.Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·www.promega.comPrinted in USA.Revised 8/09Part# TM058Page 216.D. Conditions Affecting Assay Performance (continued)Media CompositionThe Dual-Glo®Reagents have been designed to work with commonly usedculture media for mammalian cells. However, under some circumstances itmay be desirable to use alternative media formulations. Although it is difficultto predict precisely how these alternative formulations might affect theperformance of the Dual-Glo®luminescent signals, the data below can be usedas a guideline. Figure 10 shows the effect of different concentrations of severalcomponents commonly used in cell culture medium. The concentration of eachcomponent in four types of medium (RPMI 1640, DMEM, MEMα, and F12) isindicated by vertical lines on each panel in Figure 10.Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·www.promega.comPart# TM058Page 22Printed in USA.Revised 8/09A.RPMI 1640B.RPMI 1640Firefly LuciferaseRenilla LuciferaseDMEMMEMα13DMEMMEMαF12Signal Stability (Percent Remaining After 120 Minutes)Relative Luminescence (Percent)12010080604020000.112010080604020000.1F120.313100.310[CaCl2] (mM)[CaCl2] (mM)C.DMEM/MEMαRPMI 1640D.DMEM/MEMαRPMI 1640Firefly LuciferaseRenilla LuciferaseF12Relative Luminescence (Percent)12010080604020000.11Signal Stability (Percent Remaining After 120 Minutes)12010080604020000.11[CuSO4] (nM)[CuSO4] (nM)Figure 10. The effect of media composition on luminescence intensity.Purifiedfirefly or Renillaluciferase (1.67 × 10–9M, 1mg/ml gelatin) was added to a 96-wellplate at 100μl per well. Each component, noted on the X axis for each pair of graphs,was tested in Dulbecco’s PBS. In situations where the component tested was alsopart of buffered saline (i.e., NaCl), the buffered saline was adjusted to yield thedesired concentration range of the component being tested. For the phosphatetitration, 10mM HEPES was used to buffer the solution at pH 7.4. Either Dual-Glo®Luciferase Reagent (for firefly luciferase) or Dual-Glo®Luciferase Reagent plusDual-Glo®Stop & Glo®Reagent (for Renillaluciferase) was added, and luminescencemeasurements were integrated over 0.5 seconds per well. Left Panels.Luminescenceshown relative to that measured in the absence of the component. Right Panels.Signal stability in various concentrations of each component expressed as percent ofluminescence remaining after two hours. The concentration of each component inthe four types of media is indicated as a vertical line on each panel. At least threesamples were used for each data point, and all relative standard errors were <3.3%.Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·www.promega.comPrinted in USA.Revised 8/09Part# TM058Page 233628MA02_2A1010010F12100E.RPMI 1640F.RPMI 1640DMEM MEMαDMEM MEMαFirefly LuciferaseRenilla LuciferaseF1213Relative Luminescence (Percent)10080604020000.10.31310Signal Stability (Percent Remaining After 120 Minutes)120F1212010080604020000.10.310[FeSO4] (μM)G.DMEM/MEMαRPMI 1640[FeSO4] (μM)H.DMEM/MEMαRPMI 1640Firefly LuciferaseRenilla LuciferaseF12Relative Luminescence (Percent)12010080604020001Signal Stability (Percent RemainingAfter 120 Minutes)12010080604020001310F12310[KCl] (mM)[KCl] (mM)I.F12DMEM/MEMαRPMI 1640J.F12DMEM/MEMαRPMI 1640Firefly LuciferaseRenilla LuciferaseRelative Luminescence (Percent)12010080604020001310Signal Stability (Percent RemainingAfter 120 Minutes)12010080604020013103628MB02_2A0[MgSO4] (mM)[MgSO4] (mM)Figure 10 (continued).Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·www.promega.comPart# TM058Page 24Printed in USA.Revised 8/09DMEM/MEMαF12Relative Luminescence (Percent)120100806040200010Signal Stability (Percent RemainingAfter 120 Minutes)1201008060402000101001,00010,000100DMEM/MEMαF12K.RPMI 1640L.RPMI 1640Firefly LuciferaseRenilla Luciferase1,00010,000[NaCl] (mM)M.DMEM/MEMαRPMI 1640[NaCl] (mM)N.DMEM/MEMαRPMI 1640Firefly LuciferaseRenilla LuciferaseF121310F12Signal Stability (Percent RemainingAfter 120 Minutes)Relative Luminescence (Percent)12010080604020000.1112010080604020000.1310100100[ZnSO4] (μM)[ZnSO4] (μM)DMEM/MEMαF12DMEM/MEMαF12O.P.RPMI 1640Signal Stability (Percent RemainingAfter 120 Minutes)Relative Luminescence (Percent)12010080604020000.11201008060402000.11101510[PO4] (mM)[PO4] (mM)Figure 10 (continued).Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·www.promega.comPrinted in USA.Revised 8/09Part# TM058Page 253628MC02_2A0RPMI 1640Firefly LuciferaseRenilla LuciferaseTemperatureBoth firefly and Renillaluciferases are temperature sensitive, thus temperatureis an important factor in experimental precision (Figure 11). Good precision canbe achieved most easily by performing all experiments at room temperature,which is near the temperature optima for both firefly and Renillaluciferases.The assay reagents should be at room temperature before beginningmeasurements.As mentioned previously, the Dual-Glo®Buffers can be stored at roomtemperature to avoid the need for temperature equilibration before use. Theheat capacity of the substrates is low; therefore reconstitution of the Dual-Glo®Substrates with the room temperature Dual-Glo®Buffers produces reagentsthat are ready for use. If temperature equilibration is needed, incubate reagentsin a water bath at room temperature. Do not use a water bath set higher than25°C.Lower temperatures result in increased signal stabilities but lower luminescentintensities. If cold reagent is used, the luminescence will slowly increase duringthe experiment as the reagent warms. High temperatures affect firefly andRenillaluciferases differently; however, both signals become less stable. Thiscan occur if the culture plates are too warm or if the luminometer producesexcess heat within the reading chamber. As shown in Figure 11, fireflyluciferase is more sensitive to changes in temperature than Renillaluciferase.Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·www.promega.comPart# TM058Page 26Printed in USA.Revised 8/09A.B.Firefly LuciferaseRenilla Luciferase1008060402001820222426Signal Stability (Percent RemainingAfter 120 Minutes)Relative Luminescence (Percent)1008060402018202224263627MA02_2A0Temperature (°C)Temperature (°C)Figure 11. The effect of temperature on luciferase luminescence.Purified firefly orRenillaluciferase (1.67 × 10–9M, 1mg/ml gelatin) was added to a luminometer tubecontaining 100μl of Dual-Glo®Luciferase Reagent (for firefly luciferase) or Dual-Glo®Luciferase Reagent plus Dual-Glo®Stop & Glo®Reagent (for Renillaluciferase).Samples were incubated at various temperatures, and light emission was measuredon a Turner Designs Model 20e luminometer, integrated over 10 seconds after a 2-second preread delay. Panel A.Luminescence at 10 minutes is shown relative tothat measured at 22°C. Panel B.Signal stability at various temperatures is expressedas percent of luminescence remaining after 2 hours. Number of samples = 3; relativestandard error ≤3.1%.Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·www.promega.comPrinted in USA.Revised 8/09Part# TM058Page 27(a)Certain applications of this product may require licenses from others.(b)U.S. Pat. Nos. 5,283,179, 5,641,641, 5,650,289 and 5,814,471, Australian Pat. No. 649289, European Pat. No. 0 553 234 and Japanese Pat. No. 3171595 have been issued to Promega Corporation for a beetle luciferase assaymethod, which affords greater light output with improved kinetics as compared to the conventional assay.(c)U.S. Pat. No. 5,744,320, Australian Pat. No. 721172, European Pat. No. 0 833 939, Canadian Pat. No. 2,221,522,Japanese Pat. No. 3601606 and Korean Pat. No. 10-438880 have been issued to Promega Corporation for quenchingreagents and assays for enzyme-mediated luminescence.(d)The method of recombinant expression of Coleopteraluciferase is covered by U.S. Pat. Nos. 5,583,024, 5,674,713 and5,700,673.(e)This product does not convey a license to use recombinant Renillaluciferase under U.S. Pat. Nos. 5,292,658,5,418,155 and related patents. Promega sells licensed Renillaluciferase vectors, which may be used in conjunctionwith this product.© 2002, 2005, 2006, 2009 Promega Corporation. All Rights Reserved.Dual-Luciferase, QuantiLum, Steady-Glo and Stop & Glo are registered trademarks of Promega Corporation. Bright-Glo, DLR, Dual-Glo, EnduRen, GloMax, PureYield and ViviRen are trademarks of Promega Corporation.Products may be covered by pending or issued patents or may have certain limitations. Please visit our Web site formore 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 catalogfor the most up-to-date information on Promega products.Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·www.promega.comPart# TM058Page 28Printed in USA.Revised 8/09