| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 647/670 |
| MW | ~400 |
| CAS# | N/A |
| Solvent | DMSO |
| Storage | F/D/L |
| Category | EnzymeDetection HorserADIshPeroxidase(HRP) |
| Related |
| Spectrum | AdvancedSpectrumViewer |
1.PrepareAmpliteTMIRworkingsolution:
1.1 Preparea10to25mMstocksolutionofAmpliteTMIRinhigh-quality,anhydrousDMSO.Thestocksolutionshouldbeusedpromptly.Anyunusedsolutionneedtobealiquotedandrefrozenat<-20oC.
Note:Avoidrepeatedfreeze-thawcycles,andprotectfromlight.
1.2 Preparea2XAmpliteTMIRworkingsolution:Onthedayoftheexperiment,eitherdissolveAmpliteTMIRsolidinDMSOorthawanaliquotoftheAmpliteTMIRstocksolutionatroomtemperature.Preparea2Xworkingsolutionof100to250µMin50mMphosphatebufferorbufferofyourchoice,pH7with0.8units/mLperoxidase.AmpliteTMIRfinalconcentrationof50to100µMisrecommendedformeasuringH2O2concentrationinsolution.
Note:AmpliteTMIRisunstableinthepresenceofthiolssuchasDTTandb-mercaptoethanol.Thiolshigherthan10µM(finalconcentration)couldsignificantlydecreasetheassaydynamicrange.NADHandglutathione(reducedfrom:GSH)mayinterferewiththeassay.
2.RunH2O2assayinsupernatants:
2.1 Add50µLof2XAmpliteTMIRworkingsolution(fromStep1.2)intoeachwelloftheH2O2standard,blankcontrol,andtestsamplestomakethetotalH2O2assayvolumeof100µL/well.
Note:Fora384-wellplate,add25µLofsampleand25µLof2XAmpliteTMIRworkingsolutionintoeachwell.
2.2 Incubatethereactionatroomtemperaturefor0to30minutes,protectedfromlight.
2.3 MonitorthefluorescenceincreaseatEx/Em=640/680nmwithafluorescenceplatereader.
Note:Amplite™IRperoxidasesubstrateiseasytobeself-oxidized,soreadthefluorescenceassoonastheH2O2reactionmixtureisaddedtoincreasethesignaltonoiseratio.
2.4 Thefluorescenceinblankwells(withtheassaybufferonly)isusedasacontrol,andissubtractedfromthevaluesforthosewellswiththeH2O2reactions.
3.RunH2O2assayforcells:
Amplite™IRcanbeusedtomeasurethereleaseofH2O2fromcells.Thefollowingisasuggestedprotocolthatcanbemodifiedforyourspecificresearchneeds.
3.1 TheAmpliteTMIRworkingsolutionshouldbepreparedasStep1.2exceptthatthephosphatebuffershouldbereplacedwiththemediathatisusedinthecellculturesystem.Suggestedmediaincluding(a)KrebsRingersPhosphateBuffer(KRPB);(b).HanksBalancedSaltSolution(HBSS);or(c)Serum-freemedia.
3.2 Preparecellsina96-wellplate(50-100µL/well),andactivatethecellsasdesired.
Note:Thenegativecontrols(mediaaloneandnon-activatedcells)areincludedformeasuringbackgroundfluorescence.
3.3 Add50µLofH2O2reactionmixture(fromStep1.2)toeachwellofthecells,andthoseofH2O2standards.
Note:Fora384-wellplate,add25µLofcellsand25µLofH2O2reactionmixtureintoeachwell.
3.4 Incubatethereactionfor0to30minutesatroomtemperature,protectedfromlight.
3.5 MonitorthefluorescenceincreaseatEx/Em=640/680nmwithafluorescenceplatereader.
Note1:Thecontentsoftheplatecanalsobetransferredtoawhiteclearbottomplateandreadbyanabsorbancemicroplatereaderatthewavelengthof670nm.Theabsorptiondetectionhaslowersensitivitycomparedtofluorescencereading.
Note2:Thefluorescencebackgroundincreaseswithtime,thusitisimportanttosubtractthefluorescenceintensityvalueoftheblankwellsforeachdatapoint.
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