AAT Bioquest/Amplite™ Fluorimetric Hydrogen Peroxide Assay Kit *Near Infrared Fluorescence*/11502/500 Tests-免疫在线蚂蚁淘旗下平台

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商品详细AAT Bioquest/Amplite™ Fluorimetric Hydrogen Peroxide Assay Kit *Near Infrared Fluorescence*/11502/500 Tests

AAT Bioquest/Amplite™ Fluorimetric Hydrogen Peroxide Assay Kit *Near Infrared Fluorescence*/11502/500 Tests

商品编号： 11502

品牌： aatbio

市场价： ¥71060.00

美元价： 42636.00

产地：美国(厂家直采)

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产品分类：其它检测试剂盒

公司分类： Other_kits

联系Q Q： 3392242852

电话号码： 4000-520-616

电子邮箱： info@ebiomall.com

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Ex/Em(nm)	647/670
MW	N/A
CAS#	N/A
Solvent	N/A
Storage	F/D/L
Category	EnzymeDetection HorserADIshPeroxidase(HRP)
Related	ReactiveOxygenSpecies MicroplateReaders BiochemicalAssays

Hydrogenperoxide(H2O2)isareactiveoxygenmetabolicby-productthatservesasakeyregulatorforanumberofoxidativestress-relatedstates.ItisinvolvedinanumberofBIOLOGicaleventsthathavebeenlinkedtoasthma,atherosclerosis,diabeticvasculopathy,osteoporosis,anumberofneurodegenerativediseasesandDown"ssyndrome.PerhapsthemostintriguingaspectofH2O2biologyistherecentreportthatantibodieshavethecapacitytoconvertmolecularoxygenintohydrogenperoxidetocontributetothenormalrecognitionanddestructionprocessesoftheimmunesystem.Measurementofthisreactivespecieswillhelptodeterminehowoxidativestressmodulatesvariedintracellularpathways.ThisAmplite™HydrogenPeroxideAssayKitusesouruniqueAmplite™IRperoxidasesubstratetoquantifyhydrogenperoxideinsolutionsandcellextracts.Amplite™IRgeneratesthefluorescencethatispH-independentfrompH4to10.ThusitissuperioralternativetoADHP(AmplexRed™)forthedetectionsthatrequirelowpHwhereADHP(AmplexRed™)hasreducedfluorescence.Inaddition,Amplite™IRgeneratesaproductthathasmaximumabsorptionof647nmwithmaximumemissionat670nm.Thisnearinfraredabsorptionandfluorescenceminimizetheassaybackgroundthatisoftencausedbytheautofluorescenceofbiologicalsamplesthatrarelyabsorblightbeyond600nm.Itcanalsobeusedtodetectavarietyofoxidaseactivitiesthroughenzyme-coupledreactions.Thekitisanoptimized"mixandread"assaythatiscompatIBLewithHTSliquidhandlinginstruments.

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Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

1.Preparestocksolutions:

1.1 100XAmplite™IRperoxidasesubstratestocksolution:Add250µLofDMSO(ComponentE)intothevialofAmplite™IRPeroxidaseSubstrate(ComponentA).Thestocksolutionshouldbeusedpromptly;anyremainingsolutionshouldbealiquotedandrefrozenat-20^oC.

Note:Avoidrepeatedfreeze-thawcycles,andprotectfromlight.

1.2 20U/mLperoxidasestocksolution:Add1mLofAssayBuffer(ComponentC)intothevialofHorseradishPeroxidase(ComponentD).

Note:TheunusedHRPsolutionshouldbedividedintosingleusealiquotsandstoredat-20^oC.

1.3 20mMH₂O₂stocksolution:Add22.7µLof3%H₂O₂(0.88M,ComponentB)into977µLofAssayBuffer(ComponentC).

Note:ThedilutedH₂O₂stocksolutionisnotstable.Theunusedportionshouldbediscarded.

2.PrepareH₂O₂reactionmixture:

PreparetheH₂O₂reactionmixtureaccordingtothefollowingtableandkeepfromlight.

Table1.H₂O₂Reactionmixtureforone96-wellplate(2X)

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

3.PrepareserialdilutionsofH₂O₂standard(0to10μM):

3.1 Add1μLof20mMH₂O₂stocksolution(fromStep1.3)into1999μLofAssayBuffer(ComponentC)toget10μMH₂O₂standard.

3.2 Take200μLof10μMH₂O₂standardtoperform1:3serialdilutionstoget3,1,0.3,0.1,0.03,0.01and0 μMserialdilutionsofH₂O₂standard.

3.3 AddserialdilutionsofH₂O₂standardandH₂O₂-containingtestsamplesintoasolidblack96-wellmicroplateasdescribedinTables2and3.

Table2LayoutofH₂O₂standardsandtestsamplesinasolidblack96-wellmicroplate

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

Note:HS=H₂O₂Standards;BL=BlankControl;TS=TestSamples

Table3Reagentcompositionforeachwell

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

*Note:AddtheserialdilutionsofH₂O₂standardfrom0.01μMto10μMintowellsfromHS1toHS7induplicate.

4.RunH₂O₂assayinsupernatantsreaction:

4.1 Add50μLofH₂O₂reactionmixture(fromStep2)intoeachwellofH₂O₂standard,blankcontrol,andtestsamples(seeStep3.3)tomakethetotalvolumeof100µL/well.

Note:Fora384-wellplate,add25μLofsampleand25μLofH₂O₂reactionmixtureintoeachwell.

4.2 Incubatethereactionatroomtemperaturefor0to30minutes,protectedfromlight.

4.3 MonitorthefluorescenceincreasewithafluorescenceplatereaderatEx/Em=640/680nm.

Note1:Amplite™IRperoxidasesubstrateiseasytobeself-oxidized,soreadthefluorescenceassoonastheH₂O₂reactionmixturewasaddedtoincreasethesignaltonoiseratio.

Note2:Thecontentsoftheplatecanalsobetransferredtoawhiteclearbottomplateandreadbyanabsorbancemicroplatereaderatthewavelengthof650nm.Theabsorptiondetectionhaslowersensitivitycomparedtothefluorescencereading.

5.RunH₂O₂assayforcells:

TheAmplite™FluoremetricHydrogenPeroxideAssayKitcanbeusedtomeasurethereleaseofH₂O₂fromcells.Thefollowingisasuggestedprotocolthatcanbemodifiedtomeetthespecificresearchneeds.

5.1 TheH₂O₂reactionmixtureshouldbepreparedasStep2exceptthattheAssayBuffer(ComponentC)shouldbereplacedwiththemediausedinyourcellculturesystem.Suggestedmediaincluding(a)KrebsRingersPhosphateBuffer(KRPB);(b).HanksBalancedSaltSolution(HBSS);or(c)Serum-freemedia.

5.2 Preparecellsina96-wellplate(50-100μL/well),andactivatethecellsasdesired.

Note:Thenegativecontrols(mediaaloneandnon-activatedcells)areincludedformeasuringthebackgroundfluorescence.

5.3 Add50μLofH₂O₂reactionmixture(fromStep5.1)intoeachwellofcells,andH₂O₂standards(fromStep3.3).

Note:Fora384-wellplate,add25μLofcellsand25μLofH₂O₂reactionmixtureintoeachwell.

5.4 Incubatethereactionatroomtemperaturefor0to30minutes,protectedfromlight.

5.5 MonitorthefluorescenceintensitywithafluorescenceplatereaderatEx/Em=640/680nm.

References&Citations

CitationExplorer

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

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美国AAT Bioquest Inc.(前身是ABD Bioquest，Inc.)是一家为从事生命科学研究、诊断研发及药物开发的科学家研发、生产和销售生物分析研究试剂和试剂盒的公司。公司致力于光谱学检测领域，包括吸收(颜色)、荧光和发光技术。AAT Bioquest的产品帮助全世界的科学家和生物医药研究者更好的了解生物化学、免疫学、细胞生物学和分子生物学等领域。AAT Bioquest会不断研发新产品，快速地丰富各个领域的产品。 1）提供反应探针和发光探针，生物素和端粒酶能够应用于标记药物小分子和生物聚合物，如蛋白、核酸以及其他碳水化合物； 2）研究并生产荧光和发光探针用于检测蛋白，核酸和活细胞； 3）不断推出新型的荧光和发光探针用于检测多种酶，特别是检测水解酶和氧化还原酶类； 4）致力于开发用于信号转导研究的试剂； 5）提供生理和神经探针，特别是钙离子指示剂和膜电位探针。

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