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AAT Bioquest/Amplite™ Fluorimetric Xanthine Oxidase Assay Kit *Red Fluorescence*/11304/200 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(500X):Add100µLofAssayBuffer(ComponentB)intothevialofHorserADIshPeroxidase(ComponentC).
Note:TheunusedHRPsolutionshouldbedividedintosingleusealiquotsandstoredat-20°C.
1.3 2U/mLXanthineOxidase(XO)stocksolution:Add100µLofAssayBuffer(ComponentB)intothevialofXanthineOxidaseStandard(ComponentE).
Note:TheunusedXOstocksolutionshouldbedividedintosingleusealiquotsandstoredat-20°C.
2.Prepareassaymixture:
PrepareassaymixtureaccordingtoTable1andprotectfromlight.
Table1.Assaymixtureforone96-wellplate
Components | Volume |
Amplite™RedStockSolution(250X,fromStep1.1) | 20µL |
HRP(500X,fromStep1.2) | 10µL |
Xanthine(100X,ComponentD) | 50µL |
AssayBuffer(ComponentB) | 5mL |
Totalvolume | 5.08mL |
3.PrepareseriallydilutedXOstandards(0to20mU/mL):
3.1 Add10μLof2U/mLXOstocksolution(fromStep1.3)into990μLofAssayBuffer(ComponentB)toget20mU/mLXOstandardsolution.
3.2 Take300μLof20mU/mLXOstandardsolutiontoperform1:2serialdilutionstoget10,5,2.5,1.25,0.625,0.3125,0.156and0mU/mLseriallydilutedXOstandards.
3.3 AddXOstandardsand/orXO-containingtestsamplesintoasolidblack96-wellmicroplateasdescribedinTables2and3
Table2.LayoutofXOstandardsandtestsamplesinasolidblack96-wellmicroplate
BL | BL | TS | TS | …. | …. |
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XO1 | XO1 | …. | …. | …. | …. |
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XO2 | XO2 |
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XO3 | XO3 |
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XO4 | XO4 |
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XO5 | XO5 |
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XO6 | XO6 |
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XO7 | XO7 |
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Note:XO=Xanthineoxidasestandards,BL=Blankcontrol,TS=testsamples.
Table3.Reagentcompositionforeachwell
XOStandard | BlankControl | TestSample |
SerialDilutions*(50μL) | AssayBuffer(ComponentB):50μL | 50μL |
*Note1:Addtheseriallydilutedxanthineoxidasestandardsfrom0.156mU/mLto10mU/mLintoeachwellfromXO1toXO7induplicate.
Note2:HighconcentrationofXOmaycausereducedfluorescencesignalduetotheoveroxidationofAmplite™Redsubstrate(toanon-fluorescentproduct).
4.RunXOassay:
4.1 Add50μLofassaymixture(fromStep2)intoeachwelloftheXOstandards,blankcontrol,andtestsamples(seeStep3,Table2)tomakethetotalXOassayvolumeof100µL/well.
Note:Fora384-wellplate,add25μLofsampleand25μLofassaymixtureintoeachwell.
4.2 Incubatethereactionfor30to60minutesatroomtemperature,protectedfromlight.
4.3 MonitorthefluorescenceintensitywithafluorescenceplatereaderatEx/Em=530-570nm/590-600nm(optimalEx/Em=540/590nm),cutoff=570nm.
Note:Thecontentsoftheplatecanalsobetransferredtoawhiteclearbottomplateandreadbyanabsorbancemicroplatereaderatthewavelengthof576±5nm.Theabsorptiondetectionhaslowersensitivitycomparedtofluorescencereading.
| References&Citations |  CitationExplorer |
XanthineoxidoreductaseregulatesmacrophageIL1βsecretionuponNLRP3inflammasomeactivation
Authors:AnnetteIves,JohjiNomura,FABIoMartinon,ThierryRoger,DidierLeRoy,JeffreyNMiner,GregoireSimon,NathalieBusso,AlexanderSo
Journal:Naturecommunications(2015)
