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Synthesis and xanthine oxidase inhibitory activity o
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Synthesis and xanthine oxidase inhibitory activity o
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Synthesis and xanthine oxidase inhibitory activity o- Synthesis and xanthine oxidase inhibitory activity o[詳細(xì)]
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2024-09-28 01:18
應(yīng)用文章
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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(lǐng)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(lǐng):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(xì)]
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2018-11-08 10:00
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安裝說(shuō)明
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9028-79-9,半乳糖氧化酶,Galactose oxidase- 英文名稱(chēng)：GalactoseOxidasefromDactyliumdendroides；D-Galactose:oxygen6-oxidoreductaseCAS號(hào)：9028-79-9級(jí)別：Highpuritygrade分子量：68KD活力：≥30units/mgprotein活力定義：oneunitequalsachangeinabsorbanceat425nmof1.0perminuteat25℃,pH6.0usingaperosidase/o-tolidinecoupledassaywithgalactoseasthesubstrate**PH：7.0產(chǎn)品描述：Suppliedasapurifiedpowder,containingsodiumphosphateandcucroseasstablilizer.Oxdizesgalactoseandsomegalactosederivativesinbothfreeandpolymericforms.OxidationattheC6position.性狀：粉末，是由一條含639個(gè)氨基酸基團(tuán)的單聚肽鏈組成、以銅原子為活性ZX的酶。非藍(lán)氧化酶的一種，是一種含銅不顯藍(lán)色的酶。與藍(lán)銅氧化酶不一樣，在600nm附近吸收系數(shù)很小，可用電子自旋共振（ESR）譜監(jiān)測(cè)，其光譜特征與無(wú)機(jī)銅配位化合物相似用途：生化研究。催化氧化半乳糖，確定樣品中半乳糖的含量保存：-20℃[詳細(xì)]
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