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Respiration Activity MOnitoring System
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2008-05-05 00:00 873閱讀次數(shù)
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Respiration Activity MOnitoring System
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Respiration Activity MOnitoring System Respiration Activity MOnitoring System[詳細]
2008-05-05 00:00
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Cytochrome Oxidase Activity CytochromeOxidaseActivityColorimetricAssayKitrev.04/13(Catalog#K287-100;100assays;Storeat-20°C)I.Introduction:CytochromecOxidase(EC1.9.3.1)orComplexIVisthefourthcomplexoftheElectronTransportChainlocatedinthemitochondrial(orbacterial)membrane.Itprovidesenergytothecellbycoup領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領:Storekitat-20°C,protectedfromlight.WarmAssayBuffertoroomtemperaturebeforeuse.KeepEnzymeDilutionBufferonice.Readtheentireprotocolbeforeperformingtheassay.VII.ReagentPreparationandStorageConditions:DTT:Aliquotandstoreat-20°C.Thawjustbeforeuse.Cytochromec:Reconstituteeachvialwith1mlofCytochromeOxidaseAssayBuffer.Mixbyvortexingtodissolvecompletely.Add5<lofDTTsolution.Mixwellandwaitfor15min.atroomtemperature.Keepthisworkingsolutionatroomtemperature.Afterassayiscompleted,aliquotandsaverestoftheCytochromecsolutionat-20°C.ThisisnowreducedformofCytochromec.VIII.ComplexIVActivityAssayProtocol:1.EfficiencyofReductionofCytochromec:Ina96-wellplate,mix20<lofreducedCytochromecwith100<lofCytochromeOxidaseAssayBuffer.PrepareaparallelwellasblankwithonlyAssayBuffer.ReadODat550nm.TheODat550nmofreducedCytochromecisbetween0.2-0.6.Ifnot,add5<lofDTT/mlofreconstitutedCytochromecandwaitfor15min.toreadagaintheOD.2.SamplePreparation:Isolatemitochondriafromculturedcells,yeastortissuesbyusingMitochondria/CytosolFractionationKit(BVcat.#K256)orYeastMitochondriaIsolationKit(K259-50)orusecellortissuelysate(BVcat.#1067).Therecommendedrangeofpurifiedmitochondriais0.5-5<gandtissueextractis1-60<gperreaction.Dilutethetestsamples,ifneededbyEnzymeDilutionBuffer.3.Cytochromecpreparation:Prepare1:6dilutionofCytochromecbyusingpre-warmedCytochromeOxidaseAssayBuffer(onepartofCytochromecto5partsofbuffer)inaseparatetubedependingonthenumberofassaysamplesandcontrols.Prepare120<lofdilutedCytochromecperreaction.4.ComplexIVActivityAssay:Beforethereaction,setthespectrophotometerat550nmonkineticprogramfor30-45minutesat30secinterval.Addthetestsamples(approx.volume5-10<l)toeachwellofa96-wellplate.Fornegativecontrol(Blank),addequalvolumeofEnzymeDilutionBuffer.Add120<lofthedilutedCytochromecfromStep3toeachsampleandcontrolusingamultichannelpipette.ShakeandimmediatelyreadandrecorddecreaseinODoveraperiodof30-45min.Note:Therateofthereactionisrelativetoacontrolornormalsample.Therateiscalculatedinlinearrange.5.Calculations:CalculaterateofthereactionbycalculatingchangeinOD:OD/minbyusingthemaximumlinearrate.Theoxidation[詳細]
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Respiration Activity MOnitoring System
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