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AAT Bioquest/Amplite™ Fluorimetric Monoamine Oxidase Assay Kit *Red Fluorescence*/11303/200 Tests
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 571/585 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | SmallMoleculeDetection DiagnosticMolecules |
| Related | RedoxEnzymes BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
1.Preparestocksolutions:
1.1 Amplite™Redstocksolution(250X):Add40µLofDMSO(ComponentF)intothevialofAmplite™Redsubstrate(ComponentA).Thestocksolutionshouldbeusedpromptly.Anyremainingsolutionshouldbealiquotedandfrozenat-20°C.
Note1:Avoidrepeatedfreeze-thawcycles.
Note2:TheAmplite™Redsubstrateisunstableinthepresenceofthiolssuchasdithiothreitol(DTT)and2-mercaptoethanol.ThefinalconcentrationofDTTor2-mercaptoethanolinthereactionshouldbenohigherthan10μM.TheAmplite™RedsubstrateisalsounstableathighpH(>8.5).Therefore,thereactionshouldbeperformedatpH7–8.Theprovidedassaybuffer,pH7.4,isrecommended.
1.2 HRPstocksolution(200X):Add100µLofAssayBuffer(ComponentB)intothevialofhorserADIshperoxidase(ComponentC).
Note:TheunusedHRPsolutionshouldbedividedintosingleusealiquotsandstoredat-20oC.
1.3 20U/mLPlasmaAmineOxidase(PAO)stocksolution:Add125µLofAssayBuffer(ComponentB)intothevialofPlasmaAmineOxidaseStandard(ComponentE).
Note:TheunusedPAOstocksolutionshouldbedividedintosingleusealiquotsandstoredat-20oC.
2.Prepareassaymixture:
Prepareassaymixtureaccordingtothefollowingtablesandprotectfromlight.
Table1.Assaymixtureforone96-wellsolidblackplate(2X)
Components | Volume |
Amplite™RedStockSolution(250X,fromStep1.1) | 20µL |
HRPStockSolution(200X,fromStep1.2) | 25µL |
MAOSubstrate (ComponentD) | 25µL |
AssayBuffer(ComponentB) | 5mL |
Totalvolume | 5.07mL |
3.PrepareseriallydilutedPAOstandards(0to1000mU/mL):
3.1 Add50μLof20U/mLPAOstocksolution(fromStep1.3)into950μLofAssayBuffer(ComponentB)toget1000mU/mLPAOstandardsolution.
3.2 Take300μLof1000mU/mLPAOstandardsolutiontoperform1:3serialdilutionstoget300,100,30,10,3,1and0mU/mLseriallydilutedPAOstandards.
3.3 AddPAOstandardsand/orPAO-containingtestsamplesintoa96-wellsolidblackmicroplateasdescribedinTables2and3
Table2.LayoutofPAOstandardsandtestsamplesinasolidblack96-wellmicroplate
BL | BL | TS | TS | …. | …. |
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PAO1 | PAO1 | …. | …. | …. | …. |
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PAO2 | PAO2 |
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PAO3 | PAO3 |
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PAO4 | PAO4 |
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PAO5 | PAO5 |
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PAO6 | PAO6 |
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PAO7 | PAO7 |
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Note:PAO=PlasmaAmineOxidasestandards,BL=Blankcontrol,TS=testsamples.
Table3.Reagentcompositionforeachwell
PAOStandard | BlankControl | TestSample |
SerialDilutions*(50μL) | AssayBuffer(ComponentB):50μL | 50μL |
*Note1:AddtheseriallydilutedPlasmaAmineOxidasestandardsfrom1mU/mLto1000mU/mLintoeachwellfromPAO1toPAO7induplicate.
Note2:HighconcentrationofPAOmaycausereducedfluorescencesignalduetotheoveroxidationofAmplite™redsubstrate(toanon-fluorescentproduct).
4.RunPAOassay:
4.1 Add50μLofassaymixture(fromStep2)intoeachwellofthePAOstandard,blankcontrol,andtestsamples(seeStep3,Table1and2)tomakethetotalPAOassayvolumeof100µL/well.
Note:Fora384-wellplate,add25μLofsampleand25μLofassaymixtureintoeachwell.
4.2 Incubatethereactionfor30to60minutesatroomtemperature,protectedfromlight.
4.3 MonitorthefluorescenceintensitywithafluorescenceplatereaderatEx/Em=530-570/590-600nm(optimalEx/Em=540/590nm,cutoff=570nm).
Note:Thecontentsoftheplatecanalsobetransferredtoawhiteclearbottomplateandreadbyanabsorbancemicroplatereaderatthewavelengthof576±5nm.Theabsorptiondetectionhaslowersensitivitycomparedtofluorescencereading.
| References&Citations |  CitationExplorer |
AnIncreaseinPlasmaHomovanillicAcidwithCocoaExtractConsumptionIsAssociatedwiththeAlleviationofDepressiveSymptomsinOverweightorObeseAdultsonanEnergyRestrictedDietinaRandomizedControlledTrial
Authors:IdoiaIbero-Baraibar,AuroraPerez-Cornago,MariaJRamirez,JAlfredoMartínez,MAngelesZulet
Journal:TheJournalofnutrition(2016):897S--904S
