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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-6-Phosphate Dehydrogenase (G6PD) Assay Kit/13806/200 Tests
产品编号:13806
市  场 价:¥85560.00
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:$4278.00
品      牌: AAT Bioquest
公      司:AAT Bioquest
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AAT Bioquest/Amplite™ Fluorimetric Glucose-6-Phosphate Dehydrogenase (G6PD) Assay Kit/13806/200 Tests
商品介绍
Overview
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Ex/Em(nm)571/585
MWN/A
CAS#N/A
SolventN/A
StorageF/D/L
CategoryCellBIOLOGy
CellMetabolism
RelatedRedoxEnzymes
BiochemicalAssays
Glucose-6-phosphatedehydrogenase(G6PD)catalyzestheconversionofglucose-6-phosphateto6-phosphoglucono-?-lactone,thefirstandrate-limitingstepinthepentosephosphatepathway.Itiscriticalmetabolicpathwaythatsuppliesreducingenergytocells(suchaserythrocytes)bymaintainingthelevelofco-enzymenicotinamideadeninedinucleotidephosphate(NADPH),andfortheproductionofpentosesugars.TheproductionofNADPHisofgreatimportancefortissuesactivelyengagedinbiosynthesisoffattyacidsand/orisoprenoids,suchastheliver,mammaryglands,ADIposetissue,andtheadrenalglands.TheNADPHalsomaintainsthelevelofglutathioneinthesecellsthathelpsprotecttheredbloodcellsagainstoxidativedamage.DeficienciesinG6PDpredisposeindividualstonon-immunehemolyticanemia.AATBioquest"sAmplite™FluorimetricGlucose-6-PhosphateDehydrogenaseAssayKitprovidesasimple,sensitiveandrapidfluorescence-basedmethodfordetectingG6PDinbiologicalsamplessuchasserum,plasma,urine,aswellasincellculturesamples.Intheenzymecoupledassay,G6PDactivityisproportionallyrelatedtotheconcentrationofNADPHthatisspecificallymonitoredbyafluorogenicNADPHsensortoyieldahighlyredfluorescenceproduct.ThefluorescencesignalcanbereadwithafluorescencemicroplatereaderatEx/Em=540nm/590nm.WiththeG6PDassaykit,wewereabletodetectaslittleas1mU/mlG6PDina100µLreactionvolume.

Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

1.PrepareNADPstocksolution(100X):

Add100µLofH2OintothevialofNADP(ComponentC)tomake100XNADPstocksolution.

 

2.PrepareG6PDstocksolution:

Add100µLofH2Oor1xPBSbufferintothevialofG6PDStandard(ComponentD)tomake100U/mLG6PDstandardsolution.

Note:TheunusedG6PDstandardstocksolutionshouldbedividedintosingleusealiquotsandstoredat-20oC.

 

3.PrepareserialdilutionsofG6PDstandard(0to300mU/mL):

3.1   Add10μLofG6PDstocksolution(fromStep2)into990µL1xPBSbuffertogenerate1000mU/mlG6PDstandardsolution.

Note:DilutedG6PDstandardsolutionisunstable,andshouldbeusedwithin4hours.

 

3.2   Take15μLof1000mU/mlG6PDstandardsolution(fromstep3.1)into485uLof1xPBSbuffertogenerate30mU/mLG6PD,andthenperform1:3serialdilutionstoget10,3.3,1.1,0.37,0.12,0.04and0mU/mLserialdilutionsofG6PDstandard.

 

3.3   AddserialdilutionsofG6PDstandardandG6PDcontainingtestsamplesintoasolidblack96-wellmicroplateasdescribedinTables1and2.

 

Table1:LayoutofG6PDstandardsandtestsamplesinasolidblack96-wellmicroplate

BL

BL

TS

TS

….

….

 

 

 

 

 

 

G6PD1

G6PD1

….

….

….

….

 

 

 

 

 

 

G6PD2

G6PD2

 

 

 

 

 

 

 

 

 

 

G6PD3

G6PD3

 

 

 

 

 

 

 

 

 

 

G6PD4

G6PD4

 

 

 

 

 

 

 

 

 

 

G6PD5

G6PD5

 

 

 

 

 

 

 

 

 

 

G6PD6

G6PD6

 

 

 

 

 

 

 

 

 

 

G6PD7

G6PD7

 

 

 

 

 

 

 

 

 

 

               Note:G6PD=D-Glucose-6-PhosphateDehydrogenaseStandards,BL=BlankControl,TS=TestSamples.

 

Table2Reagentcompositionforeachwell

G6PDStandard

BlankControl

TestSample

SerialDilutions*:50μL

DilutionBuffer(PBS):50μL

50μL

*Note:AddtheseriallydilutedG6PDstandardsfrom0.04mU/mLto30mU/mLintowellsfromG6PD1toG6PD7induplicate.

 

4.PrepareG6PDassaymixture:

4.1   Add5mLofAssayBuffer(ComponentB)intoonebottleofEnzymeProbe(ComponentA).

 

4.2   Add50µLNADPstocksolution(100X)intothebottleofComponentA (fromStep4.1),andmixwell.

Note:ThisG6PDassaymixtureisenoughforone96-wellplate.Theunusedassaymixtureshouldbedividedintosingleusealiquotsandstoredat-20oC.

 

5.RunG6PDassay:

5.1   Add50μLofG6PDassaymixture(fromStep4.2)toeachwellofG6PDstandard,blankcontrol,andtestsamples(seeStep3.3)tomakethetotalassayvolumeof100µL/well.

Note:Fora384-wellplate,add25μLofsampleand25μLassaymixtureintoeachwell.

 

5.2   Incubatethereactionatroomtemperaturefor30minutesto2hours,protectedfromlight.

 

5.3   MonitorthefluorescenceincreasewithafluorescenceplatereaderatEx/Em=540/590nm,cutoffat570nm.

References&Citations
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1.   LegezaB,BalazsZ,NashevLG,OdermattA.(2013)Themicrosomalenzyme17beta-hydroxysteroiddehydrogenase3facesthecytoplasmandusesNADPHgeneratedbyglucose-6-phosphatedehydrogenase.Endocrinology,154,205.

2.   HeckerPA,MapangaRF,KimarCP,RibeiroRF,Jr.,BrownBH,O"ConnellKA,CoxJW,ShekarKC,AsemuG,EssopMF,StanleyWC.(2012)Effectsofglucose-6-phosphatedehydrogenasedeficiencyonthemetabolicandcardiacresponsestoobesogenicorhigh-fructosediets.AmJPhysiolEndocrinolMetab,303,E959.

3.   Al-MusawiBM,Al-AllawiN,ABDul-MajeedBA,EissaAA,JubraelJM,HamamyH.(2012)Molecularcharacterizationofglucose-6-phosphatedehydrogenasedeficientvariantsinBaghdadcity-Iraq.BMCBloodDisord,12,4.

4.   ShahSS,DiakiteSA,TraoreK,DiakiteM,KwiatkowskiDP,RockettKA,WellemsTE,FairhurstRM.(2012)Anovelcytofluorometricassayforthedetectionandquantificationofglucose-6-phosphatedehydrogenasedeficiency.SciRep,2,299.

5.   OrimadegunAE,SodeindeO.(2011)Glucose-6-phosphatedehydrogenasestatusandseverityofmalarialanaemiainNigerianchildren.JInfectDevCtries,5,792.

6.   SchneiderAM,RawatD,WeinsteinLS,GupteSA,RichardsWO.(2012)EffectsoflaparoscopicRoux-en-Ygastricbypassonglucose-6phosphatedehydrogenaseactivityinobesetype2diabetics.SurgEndosc,26,823.

7.   MillimonoTS,LouaKM,RathSL,RelvasL,BentoC,DiakiteM,JarvisM,DariesN,RibeiroLM,MancoL,KaedaJS.(2012)Highprevalenceofhemoglobindisordersandglucose-6-phosphatedehydrogenase(G6PD)deficiencyintheRepublicofGuinea(WestAfrica).Hemoglobin,36,25.

8.   GavriliukLA,KorchmaruIF,RobuMV,LysyiLT.(2011)[Glutathione-dependentenzymesandglucose-6-phosphatedehydrogenaseofbloodinpatientswithlymphosarcoma(non-Hodgkin"sdisease)].BiomedKhim,57,225.

9.   CarterN,PambaA,DuparcS,WaitumbiJN.(2011)Frequencyofglucose-6-phosphatedehydrogenasedeficiencyinmalariapatientsfromsixAfricancountriesenrolledintworandomizedanti-malarialclinicaltrials.MalarJ,10,241.

10.   EandiEberleS,GarciaRosolenN,UrtasunC,SciuccatiG,DiazL,SaviettoV,CandasA,AvalosGomezV,CervioC,BonduelM,FeliuTorresA.(2011)[Glucose6phosphatedehydrogenasedeficiency:acaseseries].ArchArgentPediatr,109,354.


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