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AAT Bioquest/Amplite™ Fluorimetric Catalase Assay Kit *Red Fluorescence*/11306/200 Tests
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 571/585 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | NeuroBIOLOGy ReactiveOxygenSpecies |
| Related | RedoxEnzymes BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
1.Preparestocksolutions:
1.1 Amplite™Redsubstratestocksolution(200X):Add65µLofDMSO(ComponentF)intothevialofAmplite™Red(ComponentA).Thestocksolutionshouldbeusedpromptly,andanyremainingsolutionneedbealiquotedandfrozenat-20°C.
Note:Avoidrepeatedfreeze-thawcycles,andprotectfromlight.
1.2 100U/mLHRPstocksolution:Add200µLofAssayBuffer(ComponentC)intothevialofHorserADIshPeroxidase(ComponentD).
Note:TheunusedHRPstocksolutionshouldbedividedintosingleusealiquotsandstoredat-20°C.
1.3 10mMH2O2stocksolution:Add10µLof3%H2O2(0.88M,ComponentB)into870µLofAssayBuffer(ComponentC).
Note:ThedilutedH2O2stocksolutionisnotstable.Theunusedportionshouldbediscarded.
2.PrepareH2O2assaybuffer:
Add5µLof10mMH2O2stocksolution(fromStep1.3)into5mLofAssayBuffer(ComponentC).
3.Prepareseriallydilutedcatalasestandards(0to2U/mL):
3.1 Add2μLof1000U/mLCatalaseStandard(ComponentE)into1000μLofAssayBuffer(ComponentC)toget2U/mLcatalasestandardsolution.
3.2 Take500μLof2U/mLcatalasestandardsolution(fromStep3.1)toperform1:2serialdilutionstoget1,0.5,0.25,0.125,0.062,0.031and0U/mLseriallydilutedcatalasestandards.
3.3 Addseriallydilutedcatalasestandardsandcatalase-containingtestsamplesintoasolidblack96-wellmicroplateasdescribedinTables2and3.
Table1.Layoutofcatalasestandardsandtestsamplesinasolidblack96-wellmicroplate
BL | BL | TS | TS | …. | …. |
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CS1 | CS1 | …. | …. | …. | …. |
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CS2 | CS2 |
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CS3 | CS3 |
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CS4 | CS4 |
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CS5 | CS5 |
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CS6 | CS6 |
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CS7 | CS7 |
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Note:CS=CatalaseStandards,BL=BlankControl,TS=TestSamples.
Table2.Reagentcompositionforeachwell
CatalaseStandards | BlankControl | TestSample |
Serialdilutions*:50μL | Assaybuffer(ComponentC):50μL | 50μL |
Note:Addtheseriallydilutedcatalasestandardsfrom0.031U/mLto2U/mLintowellsfromPS1toPS7induplicate.
4.Runcatalaseassay:
4.1 Add50μLofH2O2assaybuffer(fromStep2)intoeachwellofthecatalasestandard,blankcontrol,andtestsamples(seeStep3.3)tomakethetotalcatalaseassayvolumeof100µL/well.
Note:Fora384-wellplate,add25μLofsampleand25μLofH2O2assaybufferintoeachwell.
4.2 Incubatethereactionatroomtemperaturefor15to30minutes,protectedfromlight.
4.3 Prepareassaymixtureaccordingtothefollowingtableandkeepfromlight:
Table3.Assaymixtureforone96-wellplate
Components | Volume |
Amplite™Redsubstratestocksolution(200X,fromStep1.1) | 25μL |
HRPstocksolution(100U/mL,fromStep1.2) | 15μL |
AssayBuffer(ComponentC) | 5.0 mL |
4.4 Add50μLofassaymixture(fromStep4.3)intoeachwellofcatalasestandard,blankcontrol,andtestsamples(seeStep3.3)tomakethetotalassayvolumeof150µL/well.
Note:Fora384-wellplate,add25μLofassaymixtureintoeachwell.
4.5 Incubatethereactionatroomtemperaturefor15to30minutes,protectedfromlight.
4.6 MonitorthefluorescenceincreaseatEx/Em=540±10/590±10nm(optimalEx/Em=540/590)usingafluorescenceplatereader.
Note:Thecontentsoftheplatecanalsobetransferredintoawhiteclearbottomplateandreadbyanabsorbancemicroplatereaderatthewavelengthof576±5nm.Theabsorptiondetectionhaslowersensitivitycomparedtofluorescencereading
