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AAT Bioquest/Amplite™ Fluorimetric Goat Anti-Mouse IgG-HRP Conjugate ELISA Assay Kit *Red Fluorescence*/11540/1000 Tes
| 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-mouseIgG-HRPconjugateworkingsolution:Add2µLofgoatanti-mouseIgG-HRPconjugate(ComponentE)to10mLofPBSwith1%BSA(PBS-BSA,notincluded).
Notes1:10mLofgoatanti-mouseIgG-HRPconjugateworkingsolutionisenoughfor1plate.Theconcentrationofthisgoatanti-mouseIgG-HRPconjugateworkingsolutionisrecommendedasaninitialconcentrationtotry;
2:Theoptimalconcentrationforeachparticularapplicationshouldbedeterminedempirically.
1.3 WashtheELISAwellsthreetimeswithPBScontaining0.02%to0.05%Tween®20(PBS-Tween)anddrain.
1.4 Add100μLofthedilutedHRPconjugateworkingsolution(fromStep1.2)intoeachwell(fromStep1.3)
1.5 Incubateatroomtemperaturefor30minutes.DrainofftheHRPconjugate.
1.6 WashthewellsthreetimeswithPBS-Tweenanddrain.
2.Preparestocksolutions:
2.1 200XAmplite™Redperoxidasesubstratestocksolution:Add250µLofDMSO(ComponentD)intothevialofAmplite™RedPeroxidaseSubstrate(ComponentA).Thestocksolutionshouldbeusedpromptly,andanyremainingsolutionshouldbealiquotedandrefrozenat-20oC.
Note:50µLoftheAmplite™Redperoxidasesubstratestocksolutionisenoughfor1plate.AliquotandstoreunusedDMSOstocksolutionat‑20oC.Avoidrepeatedfreeze-thawcycles.
2.2 20mMH2O2stocksolution:Add22.7µLof3%H2O2(0.88M,ComponentB)into977µLofAssayBuffer(ComponentC).
Note:ThedilutedH2O2stocksolutionisnotstable.Theunusedportionshouldbediscarded.
3.Prepareperoxidasereactionmixture:
PreparetheperoxidasereactionmixtureaccordingtoTable1andkeepfromlight.
Table1.ProxidaseReactionMixtureforOne96-wellPlate(1X)
Components | Volume |
200XAmplite™Redperoxidasesubstratestocksolution(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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