| 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.PrepareG6Pstocksolution:
Add100µLofH2Oor1xPBSbufferintothevialofG6PStandard(ComponentD)tomake100mMG6Pstandardsolution.
Note:TheunusedG6Pstandardstocksolutionshouldbedividedintosingleusealiquotsandstoredinafreezer.
3.PrepareserialdilutionsofG6Pstandard(0to100µM):
3.1 Add10μLofG6Pstocksolution(fromStep2)into990µL1xPBSbuffertogenerate1mMG6Pstandardsolution.Andthenadd10μLof1mMG6Pstocksolutioninto990µL1xPBSbuffertogenerate100µMG6Pstandardsolution.
Note:DilutedG6Pstandardsolutionisunstable,andshouldbeusedwithin4hours.
3.2 Take200μLof1mMG6Pstandardsolutiontoperform1:3serialdilutionstoget30,10,3,1,0.3,0.1and0µMserialdilutionsofG6Pstandard.
3.3 AddserialdilutionsofG6PstandardandG6Pcontainingtestsamplesintoasolidblack96-wellmicroplateasdescribedinTables1and2.
Table1:LayoutofG6Pstandardsandtestsamplesinasolidblack96-wellmicroplate
BL | BL | TS | TS | …. | …. |
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G6P1 | G6P1 | …. | …. | …. | …. |
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G6P2 | G6P2 |
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G6P3 | G6P3 |
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G6P4 | G6P4 |
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G6P5 | G6P5 |
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G6P6 | G6P6 |
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G6P7 | G6P7 |
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Note:G6P=G6PStandards,BL=BlankControl,TS=TestSamples.
Table2Reagentcompositionforeachwell
G6PStandard | BlankControl | TestSample |
SerialDilutions*:50μL | DilutionBuffer:50μL | 50μL |
*Note:AddtheseriallydilutedG6Pstandardsfrom0.1µMto100µMintowellsfromG6P1toG6P7induplicate.
4.PrepareG6Passaymixture:
4.1 Add5mLofAssayBuffer(ComponentB)intoonebottleofEnzymeProbe(ComponentA).
4.2 Add50µLNADPstocksolution(100X,fromStep1)intothebottleofComponentA (fromStep4.1),andmixwell.
Note:ThisG6Passaymixtureisenoughforone96-wellplate.Theunusedassaymixtureshouldbedividedintosingleusealiquotsandstoredat-20oC.
5.RunG6Passay:
5.1 Add50μLofG6Passaymixture(fromStep4.2)toeachwellofG6Pstandard,blankcontrol,andtestsamples(seeStep3.3)tomakethetotalassayvolumeof100µL/well.
Note:Fora384-wellplate,add25μLofsampleand25μLassaymixtureintoeachwell.
5.2 Incubatethereactionatroomtemperaturefor30minutesto2hours,protectedfromlight.
5.3 MonitorthefluorescenceincreasewithafluorescenceplatereaderatEx/Em=530-570/590-600nm(optimalEx/Em=540/590nm,cutoffat570nm).
Note:ThecontentsoftheplatecanalsobetransferredtoawhiteclearbottomplateandreadbyabsorbancemicroplatereaderattheratioofA575nm/A605nm.TheabsorptiondetectionhaslowersensitivitycomparedtofluorescencereADIng.
| 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.
