Luciferaseisthegeneraltermgiventoaclassofoxidativeenzymesthatcatalyzereactionsthatgiveofflight,aprocessknownasbioluminescence.InBIOLOGy,researcherscantakeadvantageofthisreactionanduseitasareadoutforvariousbiologicalprocesses.Thishasperhapsbeenexploitedmostinluciferasereportercelllineswhereapromoterregionfromageneofinterestisplacedimmediatelyupstreamofthecodingsequenceforluciferase.Inthissystem,transcriptionalactivationofthegeneofinterestleadstoalevelofluciferaseexpressionthatisproportionaltothelevelofgeneactivation.
Thelevelofactivationisthenassessedbylysingthecellsandaddingtheluciferasesubstrate,D-luciferin.UsingtheOne-StepLuciferaseAssaySystemfromBPSBioscience,thisisaccomplishedinasinglestepasthelysisbufferhasbeenformulatedwithD-luciferin.Afterabriefincubationperiod,bioluminescenceisreadonaluminometer.
TheOne-StepLuciferaseAssaySystemisdesignedtobeusedforhigh-throughput,sensitivequantitationoffireflyluciferaseactivityinmammaliancellculture.Thereagentconsistsoftwocomponents,aLuciferaseReagentBuffer(ComponentA)andLuciferaseReagentSubstrate(ComponentB).ComponentAandComponentBarecombinedtoformaworkingsolutionthatcontainsallthenecessarycomponentsforcelllysisandluciferasequantitation.Thisassaysystemhasseveralfeatures:
•Sensitive–highlysensitivedetectionoffireflyluciferaseactivity.
•Stable–thesignaloutputisstableformorethantwohours,providingflexibilitywithregardtoincubationtime
•Convenient–simpleone-step,homogeneousprotocol.
•High-throughput–one-stephomogeneousprotocolminimizeshandlingstepstosupporthigh-throughputscreeningapplications
•Compatibility–workswellwithavarietyofcommonmediacontaining0-10%serumandphenolred.
•Instrumentation–doesnotrequirealuminometerwithinjectors.
•LuciferaseReagentBuffermustbeat~roomtemperature(20-25°C)beforeuse.
•LuciferaseAssayReagentshouldbeaddedtocellsforatleast5minutesbeforemeasuringluminescencetoallowcompletecelllysis.
•Formaximallightintensity,measuresampleswithin1hourofreagentaddition.
•Avoidexposingtoexcessiveheatorlightduringincubation.
•Differentcelllinesmayexhibitvariationinlysisabilityand/orluminescencesignalandmayrequireslightoptimizationbytheend-user.
•Luminescencesignalisaffectedbyassayconditions.Resultsshouldbecomparedbetweensamplesmeasuredusingthesamecelltypeandmedia/serumcombinations.
•Toanalyzemultipleplates,includeacommoncontrolsampleineachplateandnormalizetheluminescenceofeachplatetothecontrolcontainedinthesameplate.
•Backgroundluminescenceisacharacteristicofluminometerperformance,therefore,backgroundluminescencemustbesubtractedfromallreADIngsforaccuracy.
