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AAT Bioquest/Amplite™ Fluorimetric Glucose Oxidase Assay Kit *Red Fluorescence*/11300/500 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 250XAmplite™Redstocksolution:Add100µLofDMSO(ComponentE)intothevialofAmplite™Red(ComponentA).Thestocksolutionshouldbeusedpromptly.Anyremainingsolutionshouldbealiquotedandrefrozenat-20oC.
Note1:Avoidrepeatedfreeze-thawcycles.
Note2:TheAmplite™Redisunstableinthepresenceofthiolssuchasdithiothreitol(DTT)and2-mercaptoethanol.ThefinalconcentrationofDTTor2-mercaptoethanolinthereactionshouldbenohigherthan10μM.TheAmplite™RedisalsounstableathighpH(>8.5).Therefore,thereactionshouldbeperformedatpH7–8.Theprovidedassaybuffer(pH7.4)isrecommended.
1.2 50XHRPstocksolution:Add1mLofAssayBuffer(ComponentB)intothevialofHorserADIshPeroxidase(ComponentC).
Note:Theunused50XHRPstocksolutionshouldbedividedintosingleusealiquotsandstoredat-20oC.
1.3 100U/mLglucoseoxidasestocksolution:Add1mLofAssayBuffer(ComponentB)intothevialofGlucoseOxidase(ComponentD).
Note:Theunused100U/mLglucoseoxidasestocksolutionshouldbedividedintosingleusealiquotsandstoredat-20oC.
1.4 10Xglucosestocksolution:Add5mLofAssayBuffer(ComponentB)intothevialofGlucose(ComponentF).
Note:Theunused10Xglucosestocksolutionshouldbestoredat-20oC.
2.Prepareassayreactionmixture:
Prepareassayreactionmixtureaccordingtothefollowingtables,protectedfromlight.
Table1Assayreactionmixtureforone96-wellplate(2X)
Components | Volume |
250XAmplite™RedStockSolution(fromStep1.1) | 20µL |
50XHRPStockSolution(fromStep1.2) | 100µL |
10XGlucoseStockSolution(fromStep1.4) | 500µL |
AssayBuffer(ComponentB) | 4.3mL |
Totalvolume | 5mL |
Table2Layoutofglucoseoxidasestandardsandtestsamplesinasolidblack96-wellmicroplate
BL | BL | TS | TS | …. | …. |
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GOS1 | GOS1 | …. | …. | …. | …. |
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GOS2 | GOS2 |
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GOS3 | GOS3 |
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GOS4 | GOS4 |
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GOS5 | GOS5 |
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GOS6 | GOS6 |
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GOS7 | GOS7 |
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Note:GOS=GlucoseOxidaseStandards,BL=BlankControl,TS=TestSamples.
Table3.Reagentcompositionforeachwell
GlucoseOxidaseStandards | BlankControl | TestSample |
SerialDilutions*:50μL | AssayBuffer(ComponentB):50μL | 50μL |
*Note1:Addtheseriallydilutedglucoseoxidasestandardsfrom0.01to10mU/mLintoeachwellfromGOS1toGOS7induplicate.
Note2:Highconcentrationofglucoseoxidase(e.g.,100mU/mL,finalconcentration)maycausereducedfluorescencesignalduetotheoveroxidationofAmplite™Red(toanon-fluorescentproduct).
3.RunGlucoseoxidaseassay:
3.1 Prepareaglucoseoxidasestandardbydiluting2µLofthe100U/mLglucoseoxidasestocksolution(fromStep1.3)into200µLofAssayBuffer(ComponentB)tohave1000mU/mLglucoseoxidasestandardsolution.Andthentake10μLof1000mU/mLglucoseoxidasestandardsolutiontoperform1:100and1:3serialdilutionstoget10,3,1,0.3,0.1,0.03,0.01and0mU/mLseriallydilutedglucoseoxidasestandards(50μL/well).Anon-glucoseoxidasebufferisincludedasblankcontrol.Thefinalglucoseoxidaseconcentrationsshouldbetwofoldlower(i.e.,0to5mU/mL).
3.2 Add50μLofassayreactionmixture(fromStep2)intoeachwellofglucoseoxidasestandards,blankcontrol,andtestsamples(seeStep2,Table3)tomakethetotalglucoseoxidaseassayvolumeof100µL/well
Note:Fora384-wellplate,add25μLofsampleand25μLofassayreactionmixtureintoeachwell.
3.3 Incubatethereactionfor10to30minutesat37oC,protectedfromlight.
3.4 MonitorthefluorescenceintensitywithafluorescenceplatereaderatEx/Em=530-570nm/590-600nm(optimalEx/Em=540/590nm).
Note:Thecontentsoftheplatecanalsobetransferredtoawhiteclearbottomplateandreadbyanabsorbancemicroplatereaderatthewavelengthof576±5nm.Theabsorptiondetectionhaslowersensitivitycomparedtofluorescencereading.
| References&Citations |  CitationExplorer |
Areassessmentofthecarnivorousstatusofsalmonids:HepaticglucokinaseisexpressedinwildfishinKerguelenIslands
Authors:LucieMarandel,PhilippeGaudin,FrančoisGuéraud,StéphaneGlise,AlexandreHerman,ELISAbethPlagnes-Juan,VincentVéron,StéphanePanserat,JacquesLabonne
Journal:ScienceofTheTotalEnvironment(2018):276--285
Overcoming5-Furesistanceinhumannon-smallcelllungcancercellsbythecombinationof5-Fuandcisplatinthroughtheinhibitionofglucosemetabolism
Authors:Jun-gangZhao,Kai-mingRen,JunTang
Journal:TumorBIOLOGy(2014):12305--12315
