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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™ Colorimetric Peroxidase (HRP) Assay Kit *Blue Color*/11551/500 Tests
产品编号:11551
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™ Colorimetric Peroxidase (HRP) Assay Kit *Blue Color*/11551/500 Tests
商品介绍
Overview
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Ex/Em(nm)664/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.ThiskitusesAmplite™Blue,ourultrasesitivechromogenicHRPsubstrate.OurAmplite™BlueisachromogenicperoxidasesubstratethatismuchmoresensitivetobothH2O2andperoxidasethanotherchromogenicperoxidasesubstratessuchasTMB,ABTS,OPDandK-Blue.Amplite™Bluegeneratesahighlyabsorptivematerialthathasmaximumabsorptionof664nm.ThisnearinfraredabsorptionminimizesthebackgroundabsorptionthatisoftencausedbytheautoabsorptionofBIOLOGicalsamplesthatrarelyabsorblightbeyond600nm.ThekitcanbeusedforELISAs,characterizingkineticsofenzymereactionandhighthroughputscreeningofoxidaseinhibitors,etc.Thekitprovidesanoptimized"mixandread"assayprotocolthatiscompatIBLewithHTSliquidhandlinginstruments.

Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

1.Preparestocksolutions:

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

Note:Avoidrepeatedfreeze-thawcycles.

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

Note:TheunusedHRPsolutionshouldbedividedintosingleusealiquotsandstoredat-20oC.

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

Note:ThedilutedH2O2solutionisnotstable.Theunusedportionshouldbediscarded.

2.PrepareHRPreactionmixture:

PrepareHRPreactionmixtureaccordingtothefollowingtableandkeepfromlight.

Table1.HRPreactionmixtureforone96-wellplate(2X)

Components

Volume

Amplite™Blueperoxidasesubstratestocksolution(100X,fromStep1.1)

50µL

20mMH2O2stocksolution(fromStep1.3)

50µL

Assaybuffer(ComponentC)

4.9mL

Totalvolume

5mL

 

3.PrepareseriallydilutedHRPstandards(0to300mU/mL):

3.1   Add15μLof20U/mLHRPstocksolution(fromStep1.2)into985μLofAssayBuffer(ComponentC)toget300mU/mLHRPstandardsolution.

 

3.2   Take200μLof300mU/mLHRPstandardsolutiontoperform1:3serialdilutionstoget100,30,10,3,1,0.3and0mU/mLseriallydilutedHRPstandards.

 

3.3   AddHRPstandardsand/orHRP-containingtestsamplesintoawhitewall/clearbottom96-wellmicroplateasdescribedinTables2and3

Table2.LayoutofHRPstandardsandtestsamplesinawhitewall/clearbottom96-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

Table3.Reagentcompositionforeachwell:

HRPStandards

BlankControl

TestSample

SerialDilutions*:50μL

AssayBuffer(ComponentC):50μL

50μL

*Note:AddtheseriallydilutedHRPstandardsfrom0.3mU/mLto300mU/mLintowellsfromPS1toPS7induplicate.

4.RunHRPassayinsupernatants:

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

Note:Fora384-wellplate,add25μLofsampleand25μLofHRPreactionmixtureintoeachwell.

4.2   Incubatethereactionatroomtemperaturefor30to60minutes,protectedfromlight.

4.3   Monitortheabsorbancewithanabsorbanceplatereaderat664±5nm.

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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