SampleType
Freshurine,buffers,andtissueculturemedia
Excitation/Emission
660nm/690nm
MethodofAnalysis
AbsorbancePlateReader
IntendedUse
HydrogenPeroxideColorimetricDetectionBioAssay™KitallowsyoutoquantitativelymeasureH2O2inavarietyofsamples.ThisBioAssay™kitisvalidatedforuseinfreshurine,buffers,andtissueculturemedia.ThisBioAssay™kitisspeciesindependent.
TestPrinciple
In
BIOLOGicalsystems,theoneelectron(incomplete)reductionofmolecularO2duringrespirationproducesthehighlyreactivesuperoxideanionr
ADIcal(O2-),whichisknowntobethesourceofmostreactiveoxygenspecies(ROS).FurtherreductionofthereactiveO2–radicalbytheenzymesuperoxidedismutase(SOD)resultsinthecreationofH2O2.Thisisarelativelystable,(non-radical-electron-state)oxygenformwhichunlikeitsmoreoxidizedprecursor,iscapableofreadilypenetratingcellmembranebarriersandinfluencingintracellularmetabolicactivities.ManycellsproducelowlevelsofO2–andH2O2inresponsetoavarietyofextracellularstimuli,suchascytokinesorpeptidegrowthfactors.TheadditionofexogenousH2O2ortheintracellularproductioninresponsetoreceptorstimulationaffectsthefunctionofvariousproteins,includingproteinkinases,proteinphosphatases,transcriptionfactors,phospholipases,ionchannels,andGproteins.H2O2andO2mayparticipateintheproductionofsingletoxygenandperoxynitrite.Generationofthesespeciesmaybeconcurrentwithreactionsinvolvingiron,andundersomecircumstances,theymightbeimportantcontributorstoH2O2toxicity.
HydrogenPeroxide(H2O2)ColorimetricDetectionBioAssay™KitisdesignedtoquantitativelymeasureH2O2inavarietyofsamples.Ahydrogenperoxidestandardisprovidedtogenerateastandardcurve.SamplesaremixedwiththeColorimetricH2O2DetectionSubstrateandthereactionisinitiatedbyadditionofhorseradishperoxidase(HRP).HRPreactswiththesubstrateinthepresenceofhydrogenperoxidetoconvertthecolorlesssubstrateintoapinkcoloredproduct,whichisreadat560nm.IncreasingconcentrationlevelsofH2O2causeacorrespondinglinearincreaseincolor.
KitComponents
2ClearHalf-Area96-wellMicrowellPlatesHydrogenPeroxideStandard(HydrogenPeroxideat1,000uMinaspecialst
ABIlizingsolution),1x200uL5XAssayBufferConcentrate(abufferconcentratecontainingdetergentsandstabilizers),1x25mLColorimetricH2O2DetectionSubstrate(asolutionofthesubstrateinaspecialstabilizingbuffer),1x5mL50XHorseradishPeroxidaseConcentrate(aconcentratedsolutionofHRPinaspecialstabilizingsolution),1x120uL
StorageandStability
Storepowderat4°Cliquidat-20°C.Storeothercomponentsat4°C.Stablefor6monthsFormaximumrecoveryofproduct,centrifugetheoriginalvialafterthawingandpriortoremovingthecap.
References
1.Rhee,S.G.,Bae,Y.S.,Lee,S.R.&Kwon,J.Hydrogenper-oxide:akeymessengerthatmodulatesproteinphosphorylationthroughcysteineoxidation.SciSTKE2000,pe1,doi:10.1126/stke.2000.53.pe1(2000).|2.Fenton,H.J.H.LXXIII.-OxidationofTartaricAcidinthepresenceofIron.J.Chem.Soc.,Trans.,65,899-910,doi:10.1039/CT8946500899(1894)|3.Sies,H.DamagetoplasmidDNAbysingletoxygenanditsprotection.MutatRes299,183-191(1993).|4.Squadrito,G.L.&Pryor,W.A.Theformationofperoxynitriteinvivofromnitricoxideandsuperoxide.ChemBiolInteract96,203-206(1995).|5.Imlay,J.A.&Linn,S.DNAdamageandoxygenradicaltoxicity.Science240,1302-1309(1988).|6.Mello-Filho,A.C.&Meneghini,R.IronistheintracellularmetalinvolvedintheproductionofDNAdamagebyoxygenradicals.MutatRes251,109-113(1991).|7.vonSonntag,C.Thechemicalbasicofradiationbiology.238-249(Taylor&Francis,1987).|8.Henle,E.S.,Roots,R.,Holley,W.R.&Chatterjee,A.DNAstrandbreakageiscorrelatedwithunalteredbasereleaseaftergammairradiation.RadiatRes143,144-150(1995).
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