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主营:研究并生产荧光和发光探针,信号转导研究的试剂
℡ 4000-520-616
℡ 4000-520-616
AAT Bioquest/Amplite™ Luminometric Peroxidase (HRP) Assay Kit/11559/500 Tests
产品编号:11559
市  场 价:¥56560.00
场      地:美国(厂家直采)
联系QQ:1570468124
电话号码:4000-520-616
邮      箱: info@ebiomall.com
美  元  价:$2828.00
品      牌: AAT Bioquest
公      司:AAT Bioquest
公司分类:
AAT Bioquest/Amplite™ Luminometric Peroxidase (HRP) Assay Kit/11559/500 Tests
商品介绍
Overview
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Ex/Em(nm)425/None
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.

Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

1.PrepareHRPreactionmixture:

Add30μLof3%stABIlizedH2O2solution(ComponentB)into5 mLofAssayBuffer(ComponentA),andkeepfromlight.

Note:TheHRPreactionmixtureisstableatroomtemperatureforatleast8hourswithoutlooseactivityifkeptfromlight.

 

 

 

2.PrepareseriallydilutedHRPstandards(0to10mU/mL):

 

2.1   20U/mLHRPstocksolution:Add1mLofPBSwith0.1%BSAintothevialofHorseradishPeroxidase(ComponentC).

Note:TheunusedHRPstocksolutionshouldbedividedintosingleusealiquotsandstoredat-20oC.

2.2   Add1μLof20U/mLHRPstocksolution(fromStep2.1)in1999μLofPBSwith0.1%BSAtoget10mU/mLHRPstandardstocksolution.

2.3   Take200μLof10mU/mLHRPstandardstocksolutiontoperform1:2serialdilutionstoget5,2.5,1.25,0.625,0.3,0.15,0.075and0mU/mLseriallydilutedHRPstandards.

2.4   AddseriallydilutedHRPstandardsand/orHRP-containingtestsamplesintoasolidblack96-wellmicroplateasdescribedinTables1and2.

 

Table1LayoutofHRPstandardsandtestsamplesinasolidblack96-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.

Table2Reagentcompositionforeachwell

HRPStandards

BlankControl

TestSample

SerialDilutions*:50μL

PBSwith0.1%BSA:50μL

50μL

Note:AddtheseriallydilutedHRPstandardsfrom0.075mU/mLto10mU/mLintowellsfromPS1toPS7induplicate.

 

3.RunHRPassayinsupernatants:

3.1   Add50μLofHRPreactionmixture(fromStep1)intoeachwellofHRPstandard,blankcontrol,andtestsamples(seeStep2.4)tomakethetotalHRPassayvolumeof100µL/well.

Note:Fora384-wellplate,add25μLofsampleand25μLofHRPreactionmixtureintoeachwell.

 

3.2   Incubatethereactionatroomtemperaturefor30minutesto2hours,protectedfromlight.

3.3   Monitortheluminescenceintensitybyusingastandardluminometer.

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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