Overview | ![]() PrinterFriendlyVersion |
Ex/Em(nm) | 571/585 |
MW | N/A |
CAS# | N/A |
Solvent | N/A |
Storage | F/D/L |
Category | EnzymeDetection HorserADIshPeroxidase(HRP) |
Related | ReactiveOxygenSpecies MicroplateReaders BiochemicalAssays |
Spectrum | AdvancedSpectrumViewer |
1.PrepareELISAplate:
1.1 PrepareELISAmicroplate(includingappropriatecontrols):PerformallnecessaryELISApreparationsteps.
1.2 Makegoatanti-mouseIgG-HRPconjugateworkingsolution:Add2µLofgoatanti-mouseIgG-HRPconjugate(ComponentE)to10mLofPBSwith1%BSA(PBS-BSA,notincluded).
Notes1:10mLofgoatanti-mouseIgG-HRPconjugateworkingsolutionisenoughfor1plate.Theconcentrationofthisgoatanti-mouseIgG-HRPconjugateworkingsolutionisrecommendedasaninitialconcentrationtotry;
2:Theoptimalconcentrationforeachparticularapplicationshouldbedeterminedempirically.
1.3 WashtheELISAwellsthreetimeswithPBScontaining0.02%to0.05%Tween®20(PBS-Tween)anddrain.
1.4 Add100μLofthedilutedHRPconjugateworkingsolution(fromStep1.2)intoeachwell(fromStep1.3)
1.5 Incubateatroomtemperaturefor30minutes.DrainofftheHRPconjugate.
1.6 WashthewellsthreetimeswithPBS-Tweenanddrain.
2.Preparestocksolutions:
2.1 200XAmplite™Redperoxidasesubstratestocksolution:Add250µLofDMSO(ComponentD)intothevialofAmplite™RedPeroxidaseSubstrate(ComponentA).Thestocksolutionshouldbeusedpromptly,andanyremainingsolutionshouldbealiquotedandrefrozenat-20oC.
Note:50µLoftheAmplite™Redperoxidasesubstratestocksolutionisenoughfor1plate.AliquotandstoreunusedDMSOstocksolutionat‑20oC.Avoidrepeatedfreeze-thawcycles.
2.2 20mMH2O2stocksolution:Add22.7µLof3%H2O2(0.88M,ComponentB)into977µLofAssayBuffer(ComponentC).
Note:ThedilutedH2O2stocksolutionisnotstable.Theunusedportionshouldbediscarded.
3.Prepareperoxidasereactionmixture:
PreparetheperoxidasereactionmixtureaccordingtoTable1andkeepfromlight.
Table1.ProxidaseReactionMixtureforOne96-wellPlate(1X)
Components | Volume |
200XAmplite™Redperoxidasesubstratestocksolution(fromStep2.1) | 50µL |
20mMH2O2stocksolution(fromStep2.2) | 100µL |
Assaybuffer(ComponentC) | 9.85mL |
Totalvolume | 10mL |
4.RunperoxidaseassayinELISAplate:
4.1 Add100μLofperoxidasereactionmixture(fromStep3)intoeachdrainedmicroplatewellcontainingthesamplesandcontrols(fromStep1.6).
4.2 Incubatethereactionatroomtemperaturefor30minutesorlonger,protectedfromlight.
4.3 Monitorthefluorescenceincreasewithafluorescenceplatereaderatexcitation530-570nm(optimalat540nm)andemission590-600nm.
Note:Theplatecanalsobereadbyanabsorbancemicroplatereaderatthewavelengthof576±5nm.Theabsorptiondetectionhaslowersensitivitycomparedtofluorescencereading.
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
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