| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 571/585 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | CellBIOLOGy CellMetabolism |
| Related | RedoxEnzymes BiochemicalAssays |
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 | …. | …. |
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G6PD1 | G6PD1 | …. | …. | …. | …. |
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G6PD2 | G6PD2 |
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G6PD3 | G6PD3 |
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G6PD4 | G6PD4 |
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G6PD5 | G6PD5 |
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G6PD6 | G6PD6 |
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G6PD7 | G6PD7 |
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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 |  PrinterFriendlyVersion |
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.
