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AAT Bioquest/Amplite™ Fluorimetric Goat Anti-Rabbit IgG-HRP Conjugate ELISA Assay Kit *Red Fluorescence*/11541/1000 Te
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 571/585 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | EnzymeDetection HorserADIshPeroxidase(HRP) |
| Related | ReactiveOxygenSpecies MicroplateReaders BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
1.PrepareELISAplate:
1.1 PrepareELISAmicroplate(includingappropriatecontrols):PerformallnecessaryELISApreparationsteps.
1.2 Makegoatanti-rabbitIgG-HRPconjugateworkingsolution:Add2µLofgoatanti-rabbitIgG-HRPconjugate(ComponentE)into10mLofPBSwith1%BSA(PBS-BSA,notincluded).
Notes1:10mLofgoatanti-rabbitIgG-HRPconjugateworkingsolutionisenoughfor1plate.Theconcentrationofthisgoatanti-rabbitIgG-HRPconjugateworkingsolutionisrecommendedasaninitialconcentrationtotry.
2:Theoptimalconcentrationforeachparticularapplicationmayneedtobedeterminedempirically.
1.3 WashtheELISAwellsthreetimeswithPBScontaining0.02%to0.05%Tween®20(PBS-Tween)anddrain.
1.4 Add100μLofthedilutedgoatanti-rabbitIgG-HRPconjugateworkingsolution(fromStep1.2)intoeachwell(fromStep1.3)
1.5 Incubateatroomtemperaturefor30minutes.DrainofftheHRPconjugate.
1.6 WashthewellsthreetimeswithPBS-Tweenanddrain.
2.Preparestocksolutions:
2.1 200XAmplite™Redperoxidasesubstratestocksolution:Add250µLofDMSO(ComponentD)intothevialofAmplite™RedPeroxidaseSubstrate(ComponentA).The200XAmplite™Redperoxidasesubstratestocksolutionshouldbeusedpromptly.Anyremainingsolutionshouldbealiquotedandrefrozenat-20oC.
Note:50µLofthe200XAmplite™Redperoxidasesubstratestocksolutionisenoughfor1plate.Aliquotandstoreunused200XAmplite™Redperoxidasesubstratestocksolutionat‑20oC.Avoidrepeatedfreeze-thawcycles.
2.2 20mMH2O2stocksolution:Add22.7µLof3%H2O2(0.88M,ComponentB)into977µLofAssayBuffer(ComponentC).
Note:ThedilutedH2O2stocksolutionisnotstable.Theunusedportionshouldbediscarded.
3.Prepareperoxidasereactionmixture:
PrepareperoxidasereactionmixtureaccordingtoTable1andkeepfromlight.
Table1.Proxidasereactionmixtureforone96-wellplate(1X)
Components | Volume |
Amplite™Redperoxidasesubstratestocksolution(200X,fromStep2.1) | 50µL |
20mMH2O2stocksolution(fromStep2.2) | 100µL |
Assaybuffer(ComponentC) | 9.85mL |
Totalvolume | 10mL |
4.RunperoxidaseassayinELISAplate:
4.1 Add100μLofperoxidasereactionmixture(fromStep3)intoeachdrainedmicroplatewellcontainingthesamplesandcontrols(fromStep1.6).
4.2 Incubatethereactionatroomtemperaturefor30minutesorlonger,protectedfromlight.
4.3 Monitorthefluorescenceincreasewithafluorescenceplatereaderatexcitation530-570nm(optimalat540nm)andemission590-600nm.
Note:Theplatecanalsobereadbyanabsorbancemicroplatereaderatthewavelengthof576±5nm.Theabsorptiondetectionhaslowersensitivitycomparedtofluorescencereading.
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