LuciferaseReporters

Bioluminescenceassaysystemshavebecomeincreasinglyusedinbiologyandmedicalresearchlaboratoriesinadditionto(or
asalternativesto)fluorescenceandchemiluminescencedetectionstrategies.Luciferaseenzymesisolatedfromdifferentanimal
specieshaveinherentvariabilityinlightemission,allowingtwoormoreluciferaseenzymestobeusedincombinationfor
multiplexanalyses,includinginvivoimaging,cellviabilityandsingleanddualspectralluciferasereporterassays.Additionally,
luciferasereactionsareclassifiedashavingeitherflashorglowkinetics,whichhavespecificdetectionsensitivitiesand
emissiondurationtimestoaccommodatedifferentexperimentaldesigns.

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OverviewofDetectionProbes LuciferaseAssayProducts

IntroductiontoBioluminescence
Lightemissionhasbeenusedtodetectexperimentalchangesinbiologicalassaysforalmost100years(1),andwhilethelistofapplicationsthatuseemittedlightasadetectionstrategyislong,the
typeoflightemittedvaries.Luminescenceislightemissionasaresultofachemicalreactionwithouttheproductionofheatoranythermalchanges.Thistypeoflightisclearlydifferentfrom
incandescence,whichgeneratesheatandiswhyincandescentlightbulbsgethotduringuse.Luminescencecanbeseparatedintotwotypes:

Bioluminescencelightemittedfromabiologicalsource
Chemiluminescencelightemittedfromanonbiologicalsourceduetoachemicalreaction

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ChemiluminescentEnzymaticProbes

Bioluminescencevs.Fluorescence
Fluorescenceisanothertypeofemittedlightcommonlyusedinbiologicalresearchandistheproductofafluorophore,amoleculethatabsorbstheenergyfromalightsourceandthenemitslightata
differentwavelength.Bioluminescence,ontheotherhand,differsfromfluorescenceinthattheexcitationenergyissuppliedbyanenzymaticreactionratherthanfromasourceoflight.Whileboth
bioluminescenceandfluorescencearewidelyusedinscientificapplications,bioluminescentreportersdisplayanultrasensitivedetectioncapacityandhaveawiderdynamicrangecomparedto
fluorescentreportersbecauseoftheenzymaticnatureofthebioluminescentreporter.

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FluorescentProbes
Bioluminescencevs.Fluorescence.Bioluminescence(left)isemittedfromthereactionofluciferaseenzymeanditssubstrate,suchasfireflyluciferaseandluciferin,respectively.Cofactorrequirements(e.g.,ATP,O2)vary

dependingontheluciferaseused.Fluorescence(right)istheproductofafluorophore(e.g.,FITC,AlexaFluordyes)absorbingtheenergyfromalightsourceandemittingthelightenergyatadifferentwavelength.

Fluorescentreportersaresusceptibletophotobleaching,providelowquantumyieldsandhavegreaterproteinstabilityincellbasedassayscomparedtobioluminescentreporters,whichmakethem

lessamenableforuseasrealtimereporters.Cellularcomponentsalsohaveautofluorescentproperties,whichincreasesthenonspecificbackgroundanddecreasesthesensitivityoffluorescent
detectionincellbasedassays.Conversely,cellularcomponentshavenoinherentbioluminescence,allowingforgreatersensitivitywithbioluminescentassays.

However,fluorescentreportersaremoreusefulforvisualizingtargetsinlivecellsthanbioluminescentproteins,becausefluorophoresdonotrequirecofactorsorexogenoussubstratesforactivity
andaremorestablethanbioluminescentreporters.

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FluorescentLabelingReagents

BioluminescenceinNature
Usesofbioluminescenceinnaturearequitediverseandareexhibitedbybothunicellularorganismslikebacteriaanddinoflagellatesandhigherorderorganismssuchasfishandinsects.
Bioluminescenceisusedbybothterrestrialandaquaticorganisms,althoughitismorecommonlyseeninmarineanimals,especiallythoselivingintheextremedepthsoftheocean.Bioluminescence
servesmanynaturalpurposesfortheseorganisms.

Defense:Astrongbioluminescentsignalcanbeusedbypreytotemporallyblindanattackingpredator,givingthepreyachancetoescape.Thelightproducedbythepreyalsoputsthepotential

predatoratriskofbeingdetectedbyotherpredators.

Camouflage:Animalsthatnormallydwellinoceanicdarkzones,includingfireflysquids,krill,dogfishsharksandhatchetandlanternfish,cometothesurfacetofeedatnight.Theseanimalsuse
counterilluminationtoblendintosurroundingareasilluminatedbymoonlight.Similarcamouflageisalsousedbycertainspeciesofshallowwatersquidthatgiveofflighttoblendinwiththemoonlight.

Feeding:Predatorsoftenusebioluminescencetoattractprey.Theanglerfish,forexample,lurespreyusinganelongateddorsalspinethatsupportsalightproducingorganthathouses

bioluminescentbacteria.Also,someoctopi,includingStauroteuthissyrtensis,havesuckersthatarelinedwithorgansthatproducebluegreenlightthatarealsousedtoattractprey.

Mating:TerrestrialanimalssuchasfirefliesandglowwormsandmarineanimalsliketheoctopodJapetelladiaphanausebioluminescencetoattractamate.

BioluminescentReaction
Twoagentsareessentialforthebioluminescentreactiontooccur:luciferase,theenzymethatcatalyzesthereaction,andtheluciferasesubstrate.Awidevarietyofluciferaseenzymeshavebeen

discovered,althoughthegeneralmechanismofthelightproducingreactionforalloftheenzymesistheoxidativedecarboxylationoftheluciferasesubstrateinthepresenceofO 2 toyieldphoton
emission(light).

Luciferaseslikelyevolvedatmultiplestagesduringthecourseofhistorytoyieldenzymesthatdifferinexpressionpattern,substratespecificity,cofactorrequirementandenzymekinetics.Theresult

oftheseevolutionarychangesisthatsomeorganisms,suchasbacteriaandfungi,canemitlightcontinuously,whileotherorganismsemitbioluminescentflasheswithvaryingdurationandintensity.
Forexample,theluminescencefromadinoflagelleteisshort,lasting0.1seconds,whilethatfromjellyfishcanlasttensofseconds.

Thevariationinbioluminescentoutputbydifferentluciferaseenzymeshasallowedscientiststoclassifythembasedontheiroutputkineticstoaccommodatedifferentexperimentaldesigns.Luciferase

enzymeswithflashkineticshavemaximumsensitivitybecauseofhighsignalintensity,althoughtheemittedlightalsorapidlydecays.Conversely,enzymeswithglowkineticsarelesssensitivebut
stablyemitlightforatleast60minutes.

Luciferaseflashvs.glowkinetics.Schematicrepresentationofthebioluminescentoutputofluciferaseenzymeswithflashandglowkineticsasafunctionoftime.RLU:relativelightunits.

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CypridinaLuciferaseVectorsandAssays RenillaLuciferaseVectorsandAssays
 GaussiaLuciferaseVectorsandAssays FireflyLuciferaseVectorsandAssays

GaussiaDuraLuciferaseVectorsandAssays

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LuciferaseSelectionGuide

LuciferaseDetectionInstrumentationGuide

LuciferaseEnzymes
Luciferaseenzymesareactiveintheirnativestateandloseactivityuponunfoldingordenaturation,apropertythathasbeenutilizedtostudyfactorsthatinfluenceproperproteinfolding.Also,
luciferasessuchasGaussia,MetridiaandCypridinaarenaturallysecretedandthereforehavebeenusedtostudythemechanismsandregulationofthesecretorypathway.

Speciesspecificluciferasespecificity,cofactorrequirementsandphysicalcharacteristics.

Organism Luciferase Size(kDa) Substrate Requires Secreted

Photinuspyralis NorthAmericanfireflyluciferase 61 Dluciferin Mg,ATP No

Luciolacruciata Japanesefirefly(Genjibotaru)luciferase 64 Dluciferin Mg,ATP No

Luciolaitalica ItalianfireflyLuciferase 64 Dluciferin Mg,ATP No

Luciolalateralis Japanesefirefly(Heike)luciferase 64 Dluciferin Mg,ATP No

Luciolamingrelica EastEuropeanfireflyluciferase 64 Dluciferin Mg,ATP No

Photurispennsylvanica Pennsylvaniafireflyluciferase 64 Dluciferin Mg,ATP No

Pyrophorusplagiophthalamus Clickbeetleluciferase 64 Dluciferin Mg,ATP No

Phrixothrixhirtus Railroadwormluciferase 64 Dluciferin Mg,ATP No

Renillareniformis Renillaluciferase 36 Coelenterazine N/A No

Rluc8(mutantofRenillaluciferase) 36 Coelenterazine N/A No

GreenRenillaluciferase 36 Coelenterazine N/A No

Gaussiaprinceps Gaussialuciferase 20 Coelenterazine N/A Yes

GaussiaDuraluciferase 20 Coelenterazine N/A Yes

Cypridinanoctiluca Cypridinaluciferase 62 Vargulin/Cypridinaluciferin N/A Yes

Cypridinahilgendorfii Cypridina(Vargula)luciferase 62 Vargulin/Cypridinaluciferin N/A Yes

Metridialonga Metridialuciferase 23.8 Coelenterazine N/A Yes

Oplophorusgracilorostris OLuc 19 Coelenterazine N/A Yes

BioluminescentExperimentalApplications
Luciferasehasgainedtremendouspopularityinthestudybiologicalsystems,becausethebioluminescenceissensitiveandthereagentsareeasytouse.Examplesofareaswherebioluminescence
isroutinelyusedinclude:

Invivoimaging
Cellproliferationassays

Proteinfolding/secretionanalyses
Reportergeneassays

InvivoImaging
Invivoimagingisarelativelynew,noninvasivemethodthatisgainingfastacceptanceinbiomedicalresearchbecauseoftheabilitytotrackmoleculareventsinliveanimalssuchasmiceandrats.In

animaldiseasemodels,cells,pathogens,proteinsorothermoleculesarelabeledwithbioluminescentluciferasesandvisualizedafterthelocalizedorsystemicadditionofsubstrate.Lowlevellightis
abletopassthroughtissue,andthelevelofpenetrationisdependentuponthewavelengthoftheemittedlight.Therefore,althoughbioluminescenceemittedfromdeepwithinanexperimentalanimal
canbedetectedwithasensitivedetectionsystem,luciferasesthatemitlightatwavelengths>600nmhavethegreatestsensitivityforinvivoimagingapplications(2).

CellViabilityAssays
Bioluminescentassayshavealsobeendevelopedtomonitorcellviabilityandproliferation.BioluminescentcellviabilityassaysquantitatethefreeATPpresentinmetabolicallyactivecellsusingATP

dependentluciferases.Asacellpopulationproliferates,theamountofavailableATPincreases,resultinginaconcomitantincreaseinthebioluminescentsignalofthepopulation.Commonlyusednon
bioluminescentmethodstomeasurecellproliferationinclude:

QuantitationofDNAsynthesisbymeasuringtritiatedthymidine( 3 Hthymidine)orbromodeoxyuridine(BrdU)uptake
Identificationofdeadcellsusingpropidiumiodide,amembraneimpermeableDNAintercalatingdye
Quantitationofthereductionoftheintracellularenvironmentbytetrazoliumsaltreduction(MTT)

AlamarBluereductionandquantitationofintracellularATPconcentration.

ReporterGeneAssays
Reportergeneassaysareusedtoinvestigatethegeneticregulatoryelementsthatcontroltheexpressionofgenesofinterest.Ageneconsistsofmultiplefunctionalparts,includingthecodingregion
thatspecifiestheproteintobemadeandregulatoryelementsthatcontrolthetranscriptionofthecodingregion.

Thereportergeneassaycentersaroundfusingtheputativeregulatoryelementstoareportergeneandmonitoringtheamountofthereporterproteinexpressed.Becausereporterexpressionis

underthecontrolofthefusedgeneticelements,reporterexpressionisdirectlycorrelatedwiththeactivityoftheregulatoryelements.Theseelementscouldbepromoters,enhancersor5'or3'
untranslatedregions(UTRs)thatcontroleitherthetranscriptionortranslationeventsinthecell.

Thecharacteristicofgoodareporterproteinisthatitshouldbeeasytoassayordetectandisnotpresentnormallyinthetestsystem.Selectionofthereportersystemalsodependsonthe
experimentalsystem,sensitivityneedsandtheavailabledetectionstrategy,beitabsorbance,fluorescenceorbioluminescence.Theproteinsthathavebeentraditionallyusedasreportersinclude
galactosidase(lacZ),chloramphenylacetyltransferase(CAT),glucuronidase(GUS)andfluorescentproteins(green,yelloworredfluorescentprotein[GFP,YFPorRFP,respectively)and

secretoryalkalinephosphatase(SEAP).

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MammalianbetaGalactosidaseAssay

YeastbetaGalactosidaseAssay

LuciferaseReporterAssays
LuciferaseAssays
Luciferasebasedreportergeneassaysarewidelyusedbecauseoftheirultrasensitivedetectioncapacityandwidedynamicrange.Theseassaysinvolveplacingageneticregulatoryelement

upstreamofaluciferasegeneandthentransferringtheresultingreporterconstructintoanimalcells,plantcellsorbacteriathroughtransfection,transformationorinjection.Expressionofthe
luciferasereportergeneisthenmeasuredtoquantifytheactivityoftheregulatoryelement(cisacting)orproteins(transacting)inthebiologicalpathwayaffectedbythetargetelement.

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LuciferaseReporterAssays
Schematicoftheluciferasereporterassay.

Singlevs.DualSpectralAssays
Luciferaseenzymescanbeusedassinglereporterstostudyonebiologicaleventinagivenexperiment,butbecauseoftheirdifferentspectralpropertiesand/orsubstrates,multipleluciferase

enzymescanbecombinedformultiplexluciferaseexperiments.Withthesetypesofexperiments,calleddualspectralluciferasereporterassays,twoluciferaseenzymesaresimultaneously
expressedanddetectedinasinglesample.Dualspectralluciferasereporterassaysareidealfor:

Analyzingmorethanonetargetperscreen
Minimizingofftargeteffects

Identifyingcrosstalkbetweentwoormorepathways
Normalizingdatatoeliminateexperimentalartifacts

Fordatanormalizationexperiments,onereportergenerallyfunctionsasanexperimentalreporterandthesecondasaninternalcontroltoaccountfornonspecificexperimentalvariationsdueto
operatorerrororasaresultofnonspecificeffectsofbiologicalmanipulationsorcelltreatments.Additionalexperimentalvariationsinclude:

Differencesincellplating

Differencesincellviabilityorproliferationduetoanexperimentaltreatment
Edgeeffectscausedbyunevenenvironmentalconditionsinthecellcultureincubator
Nonspecificeffectsofthetreatmentcompoundonthefunctionofthereporteritself

MeasurementStrategies
Dualspectralluciferasereporterassaysusingluciferaseenzymeswithnonoverlappingluminescencespectracanbemeasuredsimultaneouslybyfilterbaseddetectioninasinglesampleusinga
singlereagent.Dualreporterassayslikethisthatuseeitherthesameordifferentsubstratesarecommerciallyavailable.

Whenusingluciferaseswithoverlappingspectra,theluciferasescanbemeasuredsequentiallyintwostepsusingtworeagentsiftheyhavedifferentsubstratespecificities.Inthetwostepmethod,
theluminescencefromthefirstluciferaseismeasured,andtheenzymeistheninactivated.Asecondreagentcontainingthesubstrateforthesecondluciferaseisthenaddedandtheactivity

measured.Theoretically,morethantwoenzymescanbemultiplexedinthiswayaslongastheyhavedifferentsubstratespecificities.

Dualspectralluciferaseassaycombinationsandmultiplexdetectionstrategies.

MultiplexDetectionStrategies

LuciferaseCombinations Substratebased Spectral/Spatialbased

NativeFirefly/Renilla Luciferin/Coelenterazine N/A

Renilla/RedFirefly Coelenterazine/Luciferin 460nmBP/615nmLP

GreenRenilla/RedFirefly Coelenterazine/Luciferin 525nmBP/640nmLP

Gaussia/Renilla Difficultbutpossiblewithcoelenterazineanalogs N/A

 Gaussia/GreenRenilla Difficultbutpossiblewithcoelenterazineanalogs N/A

Gaussia/Firefly Coelenterazine/Luciferin N/A

Gaussia/RedFirefly Coelenterazine/Luciferin 470nmBP/640nmLP

Gaussia/Cypridina Coelenterazine/Vargulin N/A

Cypridina/Renilla Vargulin/Coelenterazine N/A

Cypridina/GreenRenilla Vargulin/Coelenterazine N/A

Cypridina/RedFirefly Vargulin/Luciferin 470nmBP/640nmLP

Cypridina/Gaussia/anyFirefly Vargulin/Coelenterazine/Luciferin N/A

Cypridina/anyRenilla/Firefly Vargulin/Coelenterazine/Luciferin N/A

BP:bandpassLP:longpass

TimeCourseAssaysUsingDualspectralLuciferaseAssays
Dualspectralluciferaseassaysareidealforperformingtimecourseassaysusingasecretedluciferasetomeasuretheexperimentalpromoteractivityandanintracellularluciferaseunderthecontrol
ofahousekeepinggenepromotertomeasurecellviability.Asshowninthevideoanimationbelow,experimentalpromoteractivityresultsinluciferasesecretionintotheculturemedium,whichis
collectedatvarioustimepointsthroughouttheexperiment.Thecellsarethenlysedatthecompletionofthetimecourse,andthelysateismeasuredforintracellularluciferaseactivityasthecontrolfor

cellviability.

Notallcommerciallyavailabledualspectralluciferaseassaysarecompatiblewithtimecourseassaysbecauseoftherequirementforasecretedluciferasereporter.Forexample,Renillaluciferaseis
notsecreted,andthusthePierceRenillaFireflyLuciferaseDualReporterAssayKitwillnotworkforthistypeoftimecourseassay.However,CypridinaandGaussialuciferasesaresecreted
therefore,PierceCypridinaFireflyandGaussiaFireflydualspectralluciferasekitsarecompatiblewithtimecourseassays.

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CypridinaFireflyLuciferaseDualAssayKit

GaussiaFireflyLuciferaseDualAssayKit

References
1.KeilinD.(1966)Thehistoryofcellrespirationandcytochrome.Cambridge,:CambridgeU.P.xx,416.
2.NegrinR.S.andContagC.H.(2006)Invivoimagingusingbioluminescence:Atoolforprobinggraftversushostdisease.NatRevImmunol.6,48490.
3.SmithK.C.(1989)Thescienceofphotobiology.NewYork:PlenumPress.viii,426.
4.CampbellA.K.andHerringP.J.(1990)Imidazolopyrazinebioluminescenceincopepodsandothermarineorganisms.MarineBiology.104,21925.

5.RobisonB.H.andYoungR.E.(1981)Bioluminescenceinpelagicoctopods.PacificScience.35,3944.

ForResearchUseOnly.Notforuseindiagnosticprocedures.