| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 664/None |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | EnzymeDetection HorserADIshPeroxidase(HRP) |
| Related | ReactiveOxygenSpecies MicroplateReaders BiochemicalAssays |
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 | …. | …. |
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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
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 |  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.
