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AAT Bioquest/Amplite™ Luminometric Peroxidase (HRP) Assay Kit/11559/500 Tests
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 425/None |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | EnzymeDetection HorserADIshPeroxidase(HRP) |
| Related | ReactiveOxygenSpecies MicroplateReaders BiochemicalAssays |
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 | …. | …. |
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PS1 | PS1 | …. | …. | …. | …. |
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PS2 | PS2 |
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PS3 | PS3 |
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PS4 | PS4 |
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PS5 | PS5 |
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PS6 | PS6 |
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PS7 | PS7 |
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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 |  PrinterFriendlyVersion |
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.
