>
产品中心 >
Other_kits >
AAT Bioquest/Amplite™ Fluorimetric Peroxidase (HRP) Assay Kit *Red Fluorescence*/11552/500 Tests
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 575/590 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | EnzymeDetection HorserADIshPeroxidase(HRP) |
| Related | ReactiveOxygenSpecies MicroplateReaders BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
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 |  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.
