Cytochrome Oxidase Activity
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CytochromeOxidaseActivityColorimetricAssayKitrev.04/13(Catalog#K287-100;100assays;Storeat-20°C)I.Introduction:CytochromecOxidase(EC1.9.3.1)orComplexIVisthefourthcomplexoftheElectronTransportChainlocatedinthemitochondrial(orbacterial)membrane.Itprovidesenergytothecellbycoup领electrontransportthroughtheCytochromecchainwiththeprocessofoxidativephosphorylation.ComplexIVcontains13differentsubunitsencodedbybothmitochondrialDNAandnuclearDNA.ItreceivesanelectronfromeachofthefourCytochromecmolecules,andtransfersittooneoxygenmolecule,convertingitintotwomoleculesofwater.Inthisprocess,italsobindstofourprotonmoleculesandtranslocatesthemacrossthemembranetoestablishelectrochemicalgradient,whichisutilizedforthesynthesisofATP.CytochromeOxidaseActivityAssayKitissimple,fastandhigh-throughputadaptable.Thisassaykitcanbeusedforpurifiedmitochondriaortissueextractscontainingmitochondria.TheactivityoftheenzymeisdeterminedcolorimetricallybyfollowingtheoxidationofreducedCytochromecasanabsorbancedecreaseat550nm.Theoverallreactionisasfollows:II.Application:FastandsimplemeasurementofCytochromeOxidaseenzymaticactivityin96-wellplateformat.Mitochondrialrespirationstudies,assemblyofthecomplexes,Mitochondriaoutermembraneintegrity.III.SampleType:PurifiedmitochondriaCells/TissueextractsIV.KitContents:V.UserSuppliedReagentsandEquipment:Multi-wellspectrophotometercapableofreadingabsorbance.Multi-channelpipetVI.StorageandHand领:Storekitat-20°C,protectedfromlight.WarmAssayBuffertoroomtemperaturebeforeuse.KeepEnzymeDilutionBufferonice.Readtheentireprotocolbeforeperformingtheassay.VII.ReagentPreparationandStorageConditions:DTT:Aliquotandstoreat-20°C.Thawjustbeforeuse.Cytochromec:Reconstituteeachvialwith1mlofCytochromeOxidaseAssayBuffer.Mixbyvortexingtodissolvecompletely.Add5