Overview | PrinterFriendlyVersion |
Ex/Em(nm) | 571/585 |
MW | N/A |
CAS# | N/A |
Solvent | DMSO |
Storage | F/D/L |
Category | EnzymeDetection HorserADIshPeroxidase(HRP) |
Related | BiochemicalAssays |
Spectrum | AdvancedSpectrumViewer |
AssayProtocolforPeroxidase(HRP)withAmplite™Red(forone96wellblackplate)
A1.PrepareAmplite™Redperoxidaseworkingsolution:
1.1 Prepare250XAmplite™Redstocksolution: Add200µLofanhydrousDMSOintothevial,mixedwell.Thestocksolutionshouldbeusedpromptly.Anyunusedsolutionneedtobealiquotedandrefrozenat<-20oC.
Note:Avoidrepeatedfreeze-thawcycles,andprotectfromlight.
1.2 Prepare1XAmpliteTMRedperoxidaseworkingsolution:Onthedayoftheexperiment,eitherdissolveAmpliteTMRedsolidinDMSOorthawanaliquotoftheAmpliteTMRedstocksolutionatroomtemperature.Prepare1Xworkingsolutionbyadding20µLof250XAmpliteTMRedstocksolution(fromStep1.1)in5mLof50mMphosphatebufferorbufferofyourchoice,pH7with200µMH2O2.
Note:AmpliteTMRedisunstableinthepresenceofthiolssuchasDTTandb-mercaptoethanol.Thiolshigherthan10µM(finalconcentration)couldsignificantlydecreasetheassaydynamicrange.NADHandglutathione(reducedfrom:GSH)mayinterferewiththeassay.
A2.Runperoxidaseassayinsupernatants:
2.1 Add50µLof1XAmpliteTMRedperoxidaseworkingsolution(fromStep1.2)intoeachwelloftheperoxidase standard,blankcontrol,andtestsamplestomakethetotalperoxidaseassayvolumeof100µL/well.
Note:Fora384-wellplate,add25µLofsampleand25µLof1XAmpliteTMRedperoxidaseworkingsolutionintoeachwell.
2.2 Incubatethereactionatroomtemperaturefor10to30minutes,protectedfromlight.
2.3 MonitorthefluorescenceincreaseatEx/Em=540/590nmwithafluorescenceplatereader.
2.4 Thefluorescenceinblankwells(withtheassaybufferonly)isusedasacontrol,andissubtractedfromthevaluesforthosewellswiththeperoxidasereactions.
AssayProtocolforH2O2withAmplite™Red(forone96wellblackplate)
B1.PrepareAmpliteTMRedH2O2workingsolution:
1.1 Prepare250XAmplite™Redstocksolution:Add200µLofanhydrousDMSOintothevial,mixedwell.Thestocksolutionshouldbeusedpromptly.Anyunusedsolutionneedtobealiquotedandrefrozenat<-20oC.
Note:Avoidrepeatedfreeze-thawcycles,andprotectfromlight.
1.2 Prepare1XAmpliteTMRedH2O2workingsolution:Onthedayoftheexperiment,eitherdissolveAmpliteTMRedsolidinDMSOorthawanaliquotoftheAmpliteTMRedstocksolutionatroomtemperature.Prepare1Xworkingsolutionbyadding20µLof250Xstocksolution(fromStep1)in5mLof50mMphosphatebufferorbufferofyourchoice,pH7with0.4units/mLperoxidase.
Note:AmpliteTMRedisunstableinthepresenceofthiolssuchasDTTandb-mercaptoethanol.Thiolshigherthan10µM(finalconcentration)couldsignificantlydecreasetheassaydynamicrange.NADHandglutathione(reducedfrom:GSH)mayinterferewiththeassay.
B2.RunH2O2assayinsupernatants:
2.1 Add50µLof1XAmpliteTMRedH2O2workingsolution(fromStep1.2)intoeachwelloftheH2O2standard,blankcontrol,andtestsamplestomakethetotalH2O2assayvolumeof100µL/well.
Note:Fora384-wellplate,add25µLofsampleand25µLof1XAmpliteTMRedH2O2workingsolutionintoeachwell.
2.2 Incubatethereactionatroomtemperaturefor10to30minutes,protectedfromlight.
2.3 MonitorthefluorescenceincreaseatEx/Em=540/590nmwithafluorescenceplatereader.
2.4 Thefluorescenceinblankwells(withtheassaybufferonly)isusedasacontrol,andissubtractedfromthevaluesforthosewellswiththeH2O2reactions
References&Citations | CitationExplorer |
AssessmentofTofacitinibandRuxolitinibandtheirAntiInflammatoryEffectsonMyeloperoxidase
Authors:AmberMilton
Journal:(2017)
PatternedPhotonicNitrocelluloseforPseudo-PaperELISA
Authors:JunjieChi,BingbingGao,MiSun,FenglingZhang,EnbenSu,HongLiu,ZhongzeGu
Journal:AnalyticalChemistry(2017)
SpinalCordInflammation:MolecularImagingafterThoracicAorticIschemiaReperfusionInjury
Authors:HassanAlbadawi,JohnWChen,RahmiOklu,YueWu,GregoryWojtkiewicz,BenjaminPulli,JohnDMilner,RichardPCambria,MichaelTWatkins
Journal:Radiology(2016):152222
MyeloperoxidaseNuclearImagingforEpileptogenesis
Authors:YinianZhang,DanielPSeeburg,BenjaminPulli,GregoryRWojtkiewicz,LionelBure,WendyAtkinson,StefanSchob,YoshikoIwamoto,MuhammadAli,WeiZhang
Journal:Radiology(2015):822--830
Myeloperoxidase--Hepatocyte--StellateCellCrossTalkPromotesHepatocyteInjuryandFibrosisinExperimentalNonalcoholicSteatohepatitis
Authors:BenjaminPulli,MuhammadAli,YoshikoIwamoto,MatthiasWGZeller,StefanSchob,JennyJLinnoila,JohnWChen
Journal:Antioxidants&redoxsignaling(2015):1255--1269
Orderedcleavageofmyeloperoxidaseesterbondsreleasesactivesitehemeleadingtoinactivationofmyeloperoxidasebybenzoicacidhydrazideanalogs
Authors:JianshengHuang,ForrestSmith,PeterPanizzi
Journal:Archivesofbiochemistryandbiophysics(2014):74--85
Raisingtheshields:PCRinthepresenceofmetallicsurfacesprotectedbytailor-madecoatings
Authors:FrankDScherag,ThomasBrandstetter,JürgenRühe
Journal:ColloidsandSurfacesB:Biointerfaces(2014):576--582
MeasuringmyeloperoxidaseactivityinBIOLOGicalsamples
Authors:BenjaminPulli,MuhammadAli,RezaForghani,StefanSchob,KevinLCHsieh,GregoryWojtkiewicz,JennyJLinnoila,JohnWChen
Journal:PLoSOne(2013):e67976
Micro-volumewall-lessimmunoassaysusingpatternedplanarplates
Authors:KatherineRKozak,JianyongWang,MelvinLye,RashiTakkar,NamyongKim,HyunjaeLee,NooLiJeon,KedanLin,CrystalZhang,WaiLeeTWong
Journal:LabonaChip(2013):1342--1350
Distinguishinginflammationfromtumorandperitumoraledemabymyeloperoxidasemagneticresonanceimaging
Authors:AnneKleijn,JohnWChen,JasonSBuhrman,GregoryRWojtkiewicz,YoshikoIwamoto,MartineLLamfers,AnatOStemmer-Rachamimov,SamuelDRabkin,RalphWeissleder,RobertLMartuza
Journal:ClinicalCancerResearch(2011):4484--4493