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当前位置: 首页 > 产品中心 > Other_kits > AAT Bioquest/Amplite™ Fluorimetric Peroxidase (HRP) Assay Kit *Red Fluorescence*/11552/500 Tests
商品详细AAT Bioquest/Amplite™ Fluorimetric Peroxidase (HRP) Assay Kit *Red Fluorescence*/11552/500 Tests
AAT Bioquest/Amplite™ Fluorimetric Peroxidase (HRP) Assay Kit *Red Fluorescence*/11552/500 Tests
AAT Bioquest/Amplite™ Fluorimetric Peroxidase (HRP) Assay Kit *Red Fluorescence*/11552/500 Tests
商品编号: 11552
品牌: aatbio
市场价: ¥56560.00
美元价: 33936.00
产地: 美国(厂家直采)
公司:
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
商品介绍
Overview
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Ex/Em(nm)575/590
MWN/A
CAS#N/A
SolventN/A
StorageF/D/L
CategoryEnzymeDetection
HorserADIshPeroxidase(HRP)
RelatedReactiveOxygenSpecies
MicroplateReaders
BiochemicalAssays
Peroxidaseisasmallmolecule(MW~40KD)thatcanusuallybeconjugatedtoanantibodyina4:1ratio.Duetoitssmallsize,itrarelycausessterichindranceproblemwithantibody/antigencomplexformation.Peroxidaseisinexpensivecomparedtootherlabelingenzymes.Themajordisadvantageassociatedwithperoxidaseistheirlowtolerancetomanypreservativessuchassodiumazidethatinactivatesperoxidaseactivityevenatlowconcentration.HRPconjugatesareextensivelyusedassecondarydetectionreagentsinELISAs,immuno-histochemicaltechniquesandNorthern,SouthernandWesternblotanalyses.Weofferthisquick(10min)HRPassayinaone-step,homogeneous,nowashassaysystem.ThekitcanbeusedforELISAs,characterizingkineticsofenzymereactionandhighthroughputscreeningofoxidaseinhibitors,etc.Thekitprovidesanoptimized"mixandread"assayprotocolthatiscompatIBLewithHTSliquidhandlinginstruments.
SpectrumAdvancedSpectrumViewer

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Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

1.Preparestocksolutions:

1.1   100XAmplite™Redperoxidasesubstratestocksolution:Add250µLofDMSO(ComponentE)intothevialofAmplite™RedPeroxidaseSubstrate(ComponentA).Thestocksolutionshouldbeusedpromptly,andanyremainingsolutionshouldbealiquotedandrefrozenat-20oC.

Note:Avoidrepeatedfreeze-thawcycles,andkeepfromlight.

 

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

Note:TheunusedHRPsolutionshouldbedividedintosingleusealiquotsandstoredat-20oC.

 

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

Note:ThedilutedH2O2solutionisnotstable.Theunusedportionshouldbediscarded.

 

2.PrepareHRPreactionmixture:

PreparetheHRPreactionmixtureaccordingtothefollowingtableandkeepfromlight.

 

Table1.HRPreactionmixtureforone96-wellplate(2X)

Components

Volume

100XAmplite™Redperoxidasesubstratestocksolution(fromStep1.1)

50μL

20mMH2O2stocksolution(fromStep1.3)

50μL

AssayBuffer(ComponentC)

4.9mL

Totalvolume

5mL

 

3.PrepareseriallydilutedHRPstandards(0to10mU/mL):

3.1   Add1μLof20U/mLHRPstocksolution(fromStep1.2)in1999μLofassaybuffer(ComponentC)toget10mU/mLHRPstandardsolution.

 

3.2   Take200μLof10mU/mLHRPstandardsolutiontoperform1:3serialdilutionstoget3,1,0.3,0.1,0.03,0.01and0mU/mLseriallydilutedHRPstandards.

 

3.3   AddseriallydilutedHRPstandardsand/orperoxidase-containingtestsamplesintoasolidblack96-wellmicroplateasdescribedinTables2and3.

 

Table2.LayoutofHRPstandardsandtestsamplesinasolidblack96-wellmicroplate

BL

BL

TS

TS

….

….

 

 

 

 

 

 

PS1

PS1

….

….

….

….

 

 

 

 

 

 

PS2

PS2

 

 

 

 

 

 

 

 

 

 

PS3

PS3

 

 

 

 

 

 

 

 

 

 

PS4

PS4

 

 

 

 

 

 

 

 

 

 

PS5

PS5

 

 

 

 

 

 

 

 

 

 

PS6

PS6

 

 

 

 

 

 

 

 

 

 

PS7

PS7

 

 

 

 

 

 

 

 

 

 

       Note:PS=PeroxidaseStandards;BL=BlankControl;TS=TestSamples.

Table3Reagentcompositionforeachwell

HRPStandards

BlankControl

TestSample

SerialDilutions*:50μL

AssayBuffer(ComponentC):50μL

50μL

Note1:AddtheseriallydilutedHRPstandardsfrom0.01mU/mLto10mU/mLintowellsfromPS1toPS7induplicate.

Note2:HighlevelsofHRP(e.g.,>100mU/mLfinalconcentration)maycausereducedfluorescencesignalduetotheoveroxidationofAmplite™Red(tonon-fluorescentone).

4.RunHRPassayinsupernatants:

4.1   Add50μLofHRPreactionmixture(fromStep2)toeachwellofHRPstandard,blankcontrol,andtestsamples(seeStep3.3)tomakethetotalHRPassayvolumeof100µL/well.

Note:Fora384-wellplate,add25μLofsampleand25μLofHRPreactionmixtureintoeachwell.

 

4.2   Incubatethereactionmixtureatroomtemperaturefor15to30minutes,protectedfromlight.

 

4.3   MonitorthefluorescenceincreasewithafluorescenceplatereaderatEx/Em=540±10/590±10nm(optimalEx/Em=540/590).

Note:Thecontentsoftheplatecanalsobetransferredtoawhiteclearbottomplateandreadbyanabsorbancemicroplatereaderatthewavelengthof576±5nm.Theabsorptiondetectionhaslowersensitivitycomparedtofluorescencereading.

References&Citations
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1.   DidenkoVV,BaskinDS.(2006)Horseradishperoxidase-drivenfluorescentlabelingofnanotubeswithquantumdots.Biotechniques,40,295.

2.   AlmeidaLE,ImasatoH,TabakM.(2006)Enzymaticoxidationofdipyridamoleinhomogeneousandmicellarsolutionsinthehorseradishperoxidase-hydrogenperoxidesystem.BiochimBiophysActa,1760,216.

3.   KriegR,HalbhuberKJ.(2003)Recentadvancesincatalyticperoxidasehistochemistry.CellMolBiol(Noisy-le-grand),49,547.

4.   MatsuiT,NakayamaH,YoshidaK,ShinmyoA.(2003)VesiculartransportrouteofhorseradishC1aperoxidaseisregulatedbyN-andC-terminalpropeptidesintobaccocells.ApplMicrobiolBiotechnol,62,517.

5.   WuTP,ZhengL,RuanKC.(1998)EffectofCalciumIononConformationofHorseradishPeroxidaseIsoenzymeC.ShengWuHuaXueYuShengWuWuLiXueBao(Shanghai),30,510.

6.   GrecoO,FolkesLK,WardmanP,TozerGM,DachsGU.(2000)Developmentofanovelenzyme/prodrugcombinationforgenetherapyofcancer:horseradishperoxidase/indole-3-aceticacid.CancerGeneTher,7,1414.

7.   vanGijlswijkRP,vandeCorputMP,BezrookoveV,WiegantJ,TankeHJ,RaapAK.(2000)Synthesisandpurificationofhorseradishperoxidase-labeledoligonucleotidesfortyramide-basedfluorescenceinsituhybridization.HistochemCellBiol,113,175.

8.   VianelloF,ZennaroL,DiPaoloML,RigoA,MalacarneC,ScarpaM.(2000)Preparation,morphologicalcharacterization,andactivityofthinfilmsofhorseradishperoxidase.BiotechnolBioeng,68,488.

9.   DudkinEA,GrubergER.(1999)Relativenumberofcellsprojectingfromcontralateralandipsilateralnucleusisthmitolociintheoptictectumisdependentonvisuotopiclocation:horseradishperoxidasestudyintheleopardfrog.JCompNeurol,414,212.

10.   RotaC,ChignellCF,MasonRP.(1999)Evidenceforfreeradicalformationduringtheoxidationof2"-7"-dichlorofluorescintothefluorescentdye2"-7"-dichlorofluoresceinbyhorseradishperoxidase:possibleimplicationsforoxidativestressmeasurements.FreeRadicBiolMed,27,873.


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品牌介绍
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。
品牌分类
淋巴细胞信号传导 GPCR抗体 羰基活性生物素 细胞代谢 胺反应生物素 链霉亲和素结合物 生物素化抗IgGs 乙酰基特异性抗体 细胞骨架抗体
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