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AAT Bioquest/Amplite™ Fluorimetric Glucose Quantitation Kit/40005/500 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 | 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.
Table1Assayreactionmixtureforone96-wellplate(2X)
Components | Volume |
250XAmplite™RedStockSolution(fromStep1.1) | 20µL |
10U/mLHRPStockSolution(fromStep1.2) | 100µL |
100U/mLGlucoseOxidaseSolution(fromStep1.3) | 100µL |
AssayBuffer(ComponentB) | 4.78mL |
Totalvolume | 5mL |
Table2Layoutofglucosestandardsandtestsamplesinasolidblack96-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 |
*Note1:Addtheseriallydilutedglucosestandardsfromapproximately0.03µMto30µMintoeachwellfromGS1toGS7induplicate.
Note2:Highconcentrationofglucose(e.g.,100µµintestsampleorstandard)maycausereducedfluorescencesignalduetotheoveroxidationofAmplite™redsubstrate(toanon-fluorescentproduct).
3.RunGlucoseassay:
3.1 Prepareaglucosestandardbydilutingtheappropriateamountofthe800mMglucosestocksolution(fromStep1.4)intoassaybuffer(ComponentB)toproduceglucoseconcentrationsof30μM.Thenperform1:3serialdilutionsinassaybuffer(ComponentB)togetapproximately10,3,1,0.3,0.1and0.03μMseriallydilutedglucosestandards.Anon-glucosebuffercontrolisincludedasblankcontrol.
3.2 Add50μLofassayreactionmixture(fromStep2)intoeachwellofglucosestandard,blankcontrol,andtestsamples(seeStep2,Table3)tomakethetotalglucoseassayvolumeof100µL/well
Note:Fora384-wellplate,add25μLofsampleand25μLofassayreactionmixtureintoeachwell.
3.3 Incubatethereactionfor10to30minutesat37oC,protectedfromlight.
3.4 MonitorthefluorescenceintensitywithafluorescenceplatereaderatEx/Em=530-570nm/590-600nm(optimalEx/Em=540/590nm).
| References&Citations |  CitationExplorer |
Glucosemetabolismontogenesisinrainbowtrout(Oncorhynchusmykiss)inthelightoftherecentlysequencedgenome:newtoolsforintermediarymetabolismprogramming
Authors:LucieMarandel,VincentVéron,AnneSurget,ELISAbethPlagnes-Juan,StéphanePanserat
Journal:JournalofExperimentalBiology(2016):734--743
HighglucosepotentiatesL-FABPmediatedfibrateinductionofPPARαinmousehepatocytes
Authors:AncaDPetrescu,AveryLMcIntosh,StephenMStorey,HuanHuang,GregoryGMartin,DaniloLandrock,AnnBKier,FriedhelmSchroeder
Journal:BiochimicaetBiophysicaActa(BBA)-MolecularandCellBiologyofLipids(2013):1412--1425
