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.
COMPONENTS:
•LuciferaseReagentBuffermustbeat~roomtemperature(20-25°C)beforeuse.
•LuciferaseAssayReagentshouldbeaddedtocellsforatleast5minutesbeforemeasuringluminescencetoallowcompletecelllysis.
•Formaximallightintensity,measuresampleswithin1hourofreagentaddition.
•Avoidexposingtoexcessiveheatorlightduringincubation.
•Differentcelllinesmayexhibitvariationinlysisabilityand/orluminescencesignalandmayrequireslightoptimizationbytheend-user.
•Luminescencesignalisaffectedbyassayconditions.Resultsshouldbecomparedbetweensamplesmeasuredusingthesamecelltypeandmedia/serumcombinations.
•Toanalyzemultipleplates,includeacommoncontrolsampleineachplateandnormalizetheluminescenceofeachplatetothecontrolcontainedinthesameplate.
•Backgroundluminescenceisacharacteristicofluminometerperformance,therefore,backgroundluminescencemustbesubtractedfromallreADIngsforaccuracy.
BPS bioscience总部坐落于世界最大之一的美国圣地亚哥生命科学研究机构和公司集中地的中心。公司主要提供激酶、磷酸二酯酶、磷酸酶、组蛋白去甲基化酶、组蛋白甲基化转移酶、HDACs、聚ADP核糖聚合酶。BPS公司为药物研发用重组酶类的提供了最大选择之一。产品类型 重组酶类；分子细胞生物学；重组蛋白；分析试剂盒；底物；重组细胞系
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