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主营:研究并生产荧光和发光探针,信号转导研究的试剂
℡ 4000-520-616
℡ 4000-520-616
AAT Bioquest/Amplite™ Fluorimetric Glucose Quantitation Kit/40005/500 Tests
产品编号:40005
市  场 价:¥56560.00
场      地:美国(厂家直采)
联系QQ:1570468124
电话号码:4000-520-616
邮      箱: info@ebiomall.com
美  元  价:$2828.00
品      牌: AAT Bioquest
公      司:AAT Bioquest
公司分类:
AAT Bioquest/Amplite™ Fluorimetric Glucose Quantitation Kit/40005/500 Tests
商品介绍
Overview
PrinterFriendlyVersion

Ex/Em(nm)571/585
MWN/A
CAS#N/A
SolventN/A
StorageF/D/L
CategorySmallMoleculeDetection
DiagnosticMolecules
RelatedGlucoseTransporters
BiochemicalAssays
Glucose,amonosaccharide,isthemostimportantcarbohydrateinBIOLOGy.Itisasourceofenergyandmetabolicintermediateforcellgrowth.Glucoseisoneofthemainproductsofphotosynthesisandstartscellularrespirationinbothprokaryotesandeukaryotes.Glucoselevelisakeydiagnosticparameterformanymetabolicdisorders.Thisglucoseassaykitprovidesaquickandsensitivemethodforthemeasurementofglucoseinvariousbiologicalsamples(e.g.,serum,plasma,bodyfluid,food,growthmedium,etc.).ThekitusesourAmplite™Redsubstratethatmakingthekitrecordableinadualmore,eitherfluorimetric(Ex/Em=570nm/590nm)orcolorimetricreadout(570nm).Thekitprovidesalltheessentialcomponentswithanoptimizedassayprotocol.Theassayisrobust,andcanbereADIlyadaptedforhigh-throughputassaysinawidevarietyofapplicationsthatrequirethemeasurementofglucose.Forexample,theassaymightbesuitableformonitoringglucoselevelduringfermentationandglucosefeedinginproteinexpressionprocesses.Itmightalsobeusedformonitoringglucosetransporters.
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Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

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

….

….

 

 

 

 

 

 

GS1

GS1

….

….

….

….

 

 

 

 

 

 

GS2

GS2

 

 

 

 

 

 

 

 

 

 

GS3

GS3

 

 

 

 

 

 

 

 

 

 

GS4

GS4

 

 

 

 

 

 

 

 

 

 

GS5

GS5

 

 

 

 

 

 

 

 

 

 

GS6

GS6

 

 

 

 

 

 

 

 

 

 

GS7

GS7

 

 

 

 

 

 

 

 

 

 

              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


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