AAT Bioquest/Amplite™ Colorimetric Hydrogen Peroxide Assay Kit/11500/500 Tests,AAT Bioquest,,AAT Bioquest,aatbio,AAT Bioquest厂家直采，平行进口中国aatbio公司,,Array.Array.Array蚂蚁淘厂家直采.

产品名称：AAT Bioquest/Amplite™ Colorimetric Hydrogen Peroxide Assay Kit/11500/500 Tests

产品编号：11500

市场价：¥71060.00

场地：美国(厂家直采)

产品分类：试剂盒>其它试剂盒>其它检测试剂盒>

联系QQ：1570468124

电话号码：4000-520-616

邮箱： info@ebiomall.com

美元价：$3553.00

品牌： AAT Bioquest

公司：AAT Bioquest

公司分类：

商品介绍

Overview

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Ex/Em(nm)	650/None
MW	N/A
CAS#	N/A
Solvent	N/A
Storage	F/D/L
Category	EnzymeDetection HorserADIshPeroxidase(HRP)
Related	ReactiveOxygenSpecies MicroplateReaders

Hydrogenperoxide(H2O2)isareactiveoxygenmetabolicby-productthatservesasakeyregulatorforanumberofoxidativestress-relatedstates.ItisinvolvedinanumberofBIOLOGicaleventsthathavebeenlinkedtoasthma,atherosclerosis,diabeticvasculopathy,osteoporosis,anumberofneurodegenerativediseasesandDown"ssyndrome.PerhapsthemostintriguingaspectofH2O2biologyistherecentreportthatantibodieshavethecapacitytoconvertmolecularoxygenintohydrogenperoxidetocontributetothenormalrecognitionanddestructionprocessesoftheimmunesystem.Measurementofthisreactivespecieswillhelptodeterminehowoxidativestressmodulatesvariedintracellularpathways.ThisAmplite™ColorimetricHydrogenPeroxideAssayKitusesouruniqueAmplite™IRperoxidasesubstratetoquantifyhydrogenperoxideinsolutionsandcellextracts.UponhydrogenperoxideoxidationthecolorlessAmplite™IRgeneratesanintensebluecolorproductthatispH-independentfrompH4to10.Theexistingcolorimetrichydrogenperoxideassays(fromothervendors)oftenhaveseveresampleinterferencescausedbytheinherentabsorptionofbiologicalsamples.ThenearinfraredabsorptionofAmplite™IRproductminimizestheassaybackgroundthatsincethebiologicalsamplesrarelyabsorblightbeyond600nm.Itcanalsobeusedtodetectavarietyofoxidaseactivitiesthroughenzyme-coupledreactions.Thekitisanoptimized"mixandread"assaythatiscompatIBLewithHTSliquidhandlinginstruments.

Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

Preparestocksolutions:
1. 100XAmplite™IRperoxidasesubstratestocksolution:Add250μLofDMSO(ComponentE)intothevialofAmplite™IRPeroxidaseSubstrate(ComponentA).Thestocksolutionshouldbeusedpromptly;anyremainingsolutionshouldbealiquotedandrefrozenat-20°C.
  Note:Avoidrepeatedfreeze-thawcycles,andprotectfromlight.
2. 20U/mLperoxidasestocksolution:Add1mLofAssayBuffer(ComponentC)intothevialofHorseradishPeroxidase(ComponentD).
  Note:TheunusedHRPsolutionshouldbedividedintosingleusealiquotsandstoredat-20°C.
3. 20mMH₂O₂stocksolution:Add22.7μLof3%H₂O₂(0.88M,ComponentB)into977μLofAssayBuffer(ComponentC).
  Note:ThedilutedH₂O₂stocksolutionisnotstable.Theunusedportionshouldbediscarded.
PrepareH₂O₂reactionmixture:
PreparetheH₂O₂reactionmixtureaccordingtothefollowingtableandkeepfromlight.

Table1.H₂O₂Reactionmixtureforone96-wellplate(2X)
Components Volume
100XAmplite™IRPeroxidaseSubstrateStockSolution(fromStep1.1) 50μL
20U/mLPeroxidaseStockSolution(fromStep1.2) 200μL
AssayBuffer(ComponentC) 4.75mL
Totalvolume 5mL
PrepareserialdilutionsofH₂O₂standard(0to50μM):
Warning1:Amplite™IRPeroxidaseSubstrate(ComponentA)isunstableinthepresenceofthiolssuchasDTTandmercaptoethanol.Ifthefinalconcentrationofthethiolsishigherthan10uM,itwouldsignificantlydecreasetheassaydynamicrange.
Warning2:NADHandglutathione(reducedformofGSH)mayinterferewiththeassay.
1. Add5μLof20mMH₂O₂stocksolution(fromStep1.3)into995μLofAssayBuffer(ComponentC)toget100μMH₂O₂standard.
2. Take200μLof100μMH₂O₂standardtoperform1:2serialdilutionstoget50,25,12.5,6.25,3.125,1.1.56and0μMserialdilutionsofH₂O₂standard.
3. AddserialdilutionsofH₂O₂standardandH₂O₂-containingtestsamplesintoawhitewall/clearbottom96-wellmicroplateasdescribedinTables2and3.
Table2.LayoutofH₂O₂standardsandtestsamplesinawhitewall/clearbottom96-wellmicroplate
BL BL TS TS .... ....
HS1 HS1 .... .... .... ....
HS2 HS2
HS3 HS3
HS4 HS4
HS5 HS5
HS6 HS6
HS7 HS7

Table3.Reagentcompositionforeachwell
H₂O₂Standard BlankControl TestSample
SerialDilutions*:50μL AssayBuffer(ComponentC):50μL 50μL
*Note:AddtheserialdilutedofH₂O₂standardfrom1.56μMto50μMintowellsfromHS1toHS7induplicate.
RunH₂O₂assayinsupernatantsreaction:
1. Add50μLofH₂O₂reactionmixture(fromStep2)intoeachwellofH₂O₂standard,blankcontrol,andtestsamples(seeStep3.3)tomakethetotalvolumeof100μL/well.
  Note:Fora384-wellplate,add25μLofsampleand25μLofH₂O₂reactionmixtureintoeachwell.
2. Incubatethereactionatroomtemperaturefor10to30minutes,protectedfromlight.
3. Monitortheabsorbancewithanabsorbanceplatereaderat650nm.
RunH₂O₂assayforcells:
TheAmplite™ColorimetricHydrogenPeroxideAssayKitcanbeusedtomeasurethereleaseofH₂O₂fromcells.Thefollowingisasuggestedprotocolthatcanbemodifiedtomeetthespecificresearchneeds.
1. TheH₂O₂reactionmixtureshouldbepreparedasStep2exceptthattheAssayBuffer(ComponentC)shouldbereplacedwiththemediausedinyourcellculturesystem.Suggestedmediaincluding(a)KrebsRingersPhosphateBuffer(KRPB);(b).HanksBalancedSaltSolution(HBSS);or(c)Serum-freemedia.
2. Preparecellsina96-wellplate(50-100μL/well),andactivatethecellsasdesired.
  Note:Thenegativecontrols(mediaaloneandnon-activatedcells)areincludedformeasuringthebackgroundfluorescence.
3. Add50μLofH₂O₂reactionmixture(fromStep5.1)intoeachwellofcells,andH₂O₂standards(fromStep3.3).
  Note:Fora384-wellplate,add25μLofcellsand25μLofH₂O₂reactionmixtureintoeachwell.
4. Incubatethereactionatroomtemperaturefor10to60minutes,protectedfromlight.
5. Monitortheabsorbancewithanabsorbanceplatereaderat650nm.

References&Citations

CitationExplorer

Plasma-activatedmediumselectivelyeliminatesundifferentiatedhumaninducedpluripotentstemcells
Authors:RyoMatsumoto,KazunoriShimizu,TakunoriNagashima,HiromasaTanaka,MasaakiMizuno,FumitakaKikkawa,MasaruHori,HiroyukiHonda
Journal:RegenerativeTherapy(2016):55--63

品牌介绍

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问答列表

Components	Volume
100XAmplite™IRPeroxidaseSubstrateStockSolution(fromStep1.1)	50μL
20U/mLPeroxidaseStockSolution(fromStep1.2)	200μL
AssayBuffer(ComponentC)	4.75mL
Totalvolume	5mL

BL	BL	TS	TS	....	....
HS1	HS1	....	....	....	....
HS2	HS2
HS3	HS3
HS4	HS4
HS5	HS5
HS6	HS6
HS7	HS7

H₂O₂Standard	BlankControl	TestSample
SerialDilutions*:50μL	AssayBuffer(ComponentC):50μL	50μL