| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 575/None |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | SmallMoleculeDetection DiagnosticMolecules |
| Related | GlucoseTransporters BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
1.Preparestocksolutions:
1.1 250XAmplite™Redstocksolution:Add100µLofDMSO(ComponentE)intothevialofAmplite™Redsubstrate(ComponentA).Thestocksolutionshouldbeusedpromptly.Anyremainingsolutionshouldbealiquotedandrefrozenat-20oC.
Note1:Avoidrepeatedfreeze-thawcycles.
Note2:TheAmplite™Redsubstrateisunstableinthepresenceofthiolssuchasdithiothreitol(DTT)and2-mercaptoethanol.ThefinalconcentrationofDTTor2-mercaptoethanolinthereactionshouldbenohigherthan10μM.TheAmplite™RedsubstrateisalsounstableathighpH(>8.5).Therefore,thereactionshouldbeperformedatpH7–8.Theprovidedassaybuffer(pH7.4)isrecommended.
1.2 10U/mLHRPstocksolution:Add1mLofassaybuffer(ComponentB)intothevialofhorseradishperoxidase(ComponentC).
Note:TheunusedHRPsolutionshouldbedividedintosingleusealiquotsandstoredat-20oC.
1.3 100U/mLglucoseoxidasesolution:Add1mLofassaybuffer(ComponentB)intothevialofglucoseoxidase(ComponentD).
Note:Theunusedglucoseoxidasesolutionshouldbedividedintosingleusealiquotsandstoredat-20oC.
1.4 800mMglucosestocksolution:Add1mLofassaybuffer(ComponentB)intothevialofglucose(ComponentF).
Note:Theunusedglucosesolutionshouldbestoredat-20oC.
2.Prepareassayreactionmixture:
PrepareAssayreactionmixtureaccordingtothefollowingtables,protectedfromlight.
Table1Assayreactionmixtureforoneclearbottom96-wellmicroplate(2X)
Components | Volume |
250XAmplite™RedStockSolution(fromStep1.1) | 20μL |
10U/mLHRPStockSolution(fromStep1.2) | 100μL |
100U/mLGlucoseOxidaseSolution(fromStep1.3) | 100μL |
AssayBuffer(ComponentB) | 4.78mL |
Totalvolume | 5mL |
Table2Layoutofglucosestandardsandtestsamplesinaclearbottom96-wellmicroplate
BL | BL | TS | TS | …. | …. |
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GS1 | GS1 | …. | …. | …. | …. |
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GS2 | GS2 |
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GS3 | GS3 |
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GS4 | GS4 |
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GS5 | GS5 |
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GS6 | GS6 |
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GS7 | GS7 |
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Note:GS=Glucosestandards,BL=Blankcontrol,TS=testsamples.
Table3.Reagentcompositionforeachwell
GlucoseStandard | BlankControl | TestSample |
SerialDilutions*:50μL | AssayBuffer(ComponentB):50μL | 50μL |
*Note:Addtheseriallydilutedglucosestandardsfromapproximately1.6µMto100µMintoeachwellfromGS1toGS7induplicate.
3.RunGlucoseassay:
3.1 Prepareaglucosestandardbydilutingtheappropriateamountofthe800mMglucosestocksolution(fromStep1.4)intoassaybuffer(ComponentB)toproduceglucoseconcentrationsof100μM.Thenperform1:2serialdilutionsinassaybuffer(ComponentB)togetapproximately50,25,12.5,6.3,3.1and1.6μMseriallydilutedglucosestandards.Anon-glucosebuffercontrolisincludedasblankcontrol.
3.2 Add50μLofassayreactionmixture(fromStep2)intoeachwellofglucosestandard,blankcontrol,andtestsamples(seeStep2,Table3)tomakethetotalglucoseassayvolumeof100µL/well
Note:Fora384-wellplate,add25μLofsampleand25μLofassayreactionmixtureintoeachwell.
3.3 Incubatethereactionfor10to30minutesat37oC,protectedfromlight.
3.4 MonitortheabsorbanceincreasewithanabsorbanceplatereaderatOD=570nm.
| References&Citations |  CitationExplorer |
MicroRNAsregulategeneplasticityduringcoldshockinzebrafishlarvae
Authors:I-ChenHung,Yu-ChuanHsiao,HSunnySun,Tsung-MingChen,Shyh-JyeLee
Journal:BMCgenomics(2016):922
Glucosemetabolismandgeneexpressioninjuvenilezebrafish(Daniorerio)challengedwithahighcarbohydratediet:effectsofanacuteglucosestimulusduringlateembryoniclife
Authors:FilipaRocha,JorgeDias,SofiaEngrola,PauloGavaia,IngeGeurden,MariaTeresaDinis,StephanePanserat
Journal:BritishJournalofNutrition(2015):403--413
Glucoseoverloadinyolkhaslittleeffectonthelong-termmodulationofcarbohydratemetabolicgenesinzebrafish(Daniorerio)
Authors:FilipaRocha,JorgeDias,SofiaEngrola,PauloGavaia,IngeGeurden,MariaTeresaDinis,StephanePanserat
Journal:JournalofExperimentalBiology(2014):1139--1149
ImpactofL-FABPandglucoseonpolyunsaturatedfattyacidinductionofPPARα-regulatedβ-oxidativeenzymes
Authors:AncaDPetrescu,HuanHuang,GregoryGMartin,AveryLMcIntosh,StephenMStorey,DaniloLandrock,AnnBKier,FriedhelmSchroeder
Journal:AmericanJournalofPhysiology-GastrointestinalandLiverPhysiology(2013):G241--G256
InhibitorsoffattyacidsynthesisinducePPARα-regulatedfattyacidβ-oxidativegenes:synergisticrolesofL-FABPandglucose
Authors:HuanHuang,AveryLMcIntosh,GregoryGMartin,AncaDPetrescu,KerstinKLandrock,DaniloLandrock,AnnBKier,FriedhelmSchroeder
Journal:PPARresearch(2013)
Aquaporin-9proteinistheprimaryrouteofhepatocyteglyceroluptakeforglycerolgluconeogenesisinmice
Authors:SABInaJelen,SörenWacker,CamiloAponte-Santamaría,MartinSkott,AleksandraRojek,UrbanJohanson,PerKjellbom,SørenNielsen,BertLdeGroot,MichaelRützler
Journal:JournalofBiologicalChemistry(2011):44319--44325
