| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 420/480 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | EnzymeDetection HorserADIshPeroxidase(HRP) |
| Related | ReactiveOxygenSpecies MicroplateReaders BiochemicalAssays |
1.PrepareGlutathionePeroxidase(GPx)standardstocksolution:
Add50µLofddH2Oor1×PBSbufferintothevialofGPxstandard(ComponentA)tomake10U/mLstandardstocksolution.
Note:TheunusedGPxstandardstocksolutionshouldbedividedintosingleusealiquotsandstoredat-20ºC.
2.PrepareserialdilutionsofGPxstandard:
2.1 Add4μLofGPxstandardstocksolution(10U/mL,fromStep1)into996µL1×PBSbuffertogeneratestandardsolutionattheconcentrationof40mU/mL.
Note:DilutedGPxstandardsolutionisunstable,andshouldbeusedwithin4hours.
2.2 Take200μLof40mU/mLGPxstandardsolutiontoperform1:2serialdilutionstogetapproximately20,10,5,2.5,1.25,0.625and0mU/mLserialdilutionsofGPxstandard.
2.3 AddserialdilutionsofGPxstandardandGPxcontainingtestsamplesintoasolidblack96-wellmicroplateasdescribedinTables1and2.
Table1LayoutofGPxstandardsandtestsamplesinasolidblack96-wellmicroplate
BL | BL | TS | TS | …. | …. |
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GP1 | GP1 | …. | …. | …. | …. |
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GP2 | GP2 |
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GP3 | GP3 |
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GP4 | GP4 |
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GP5 | GP5 |
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GP6 | GP6 |
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GP7 | GP7 |
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Note:GP=GPxStandards,BL=BlankControl,TS=TestSamples.
Table2Reagentcompositionforeachwell
GPxStandard | BlankControl | TestSample |
SerialDilutions*:50μL | 1×PBSBuffer:50μL | 50μL |
*Note:AddtheseriallydilutedGPxstandardsfromapproximately0.6mU/mLto40mU/mLintowellsfromGP1toGP7induplicate.
3.PrepareGSHstocksolution(100X):
Add100µLofddH2OintothevialofGSH(ComponentD)tomake100XGSHstocksolution.
4.PrepareGPxsubstratestocksolution(100X):
Make100µLofddH2Ointothevialofsubstrate(ComponentE)tomake100Xsubstratestocksolution.
5.PrepareGPxassaymixture:
5.1 Add5mLofAssayBuffer(ComponentB)intoabottleofEnzymeMix(ComponentC).
5.2 Add50µLGSHstocksolution(ComponentD,fromStep3),50µLsubstratestocksolution(ComponentE,fromStep4)intothebottleofComponentB+C(fromStep5.1),andmixwelltomakeGPxassaymixture(ComponentB+C+D+E).
Note1:ThisGPxassaymixtureisenoughforone96-wellplate.Itisnotstable,pleaseuseitpromptly.
Note2:ItisnotrecommendstoringunusedGPxassaymixture.OnemightdividetheunusedComponentsB+Cmixture(fromStep5.1)intosingleusealiquotsandstoredat-20ºCalthoughthesensitivitymightdecrease.
Note3;Divideunused100XGSHstocksolution(fromstep3),and100XGPxsubstratestocksolution(fromstep4)intosingleusealiquotsandstoredat-20ºC.
6.RunGPxassay:
6.1 Add50μLofGPxassaymixture(fromStep5.2)toeachwellofGPxstandard,blankcontrol,andtestsamples(seeStep2.3)tomakethetotalvolumeof100µL/well.
Note:Fora384-wellplate,add25μLofsampleand25μLofGPxassaymixtureintoeachwell.
6.2 Incubatethereactionatroomtemperaturefor30minutes,protectedfromlight.
7.RunNADPassay:
7.1Add20μLQuestFluor™NADPProbe(ComponentF)intoeachwellofGPxstandard,blankcontrol,andtestsamples,mixwell.
7.2Add20μLNADPAssaySolution(ComponentG)intoeachwell,mixwell.
Note:Fora384-wellplate,add25μLofsampleand10μLofQuestFluor™NADPProbe(ComponentF)and10μLNADPAssaySolution(ComponentG)intoeachwell.
7.3Incubatethereactionatroomtemperaturefor10-20minutes,protectedfromlight.
7.4Add15μLEnhancer(ComponentH)toeachwelltomakethetotalassayvolumeof155μL/well,andincubateatroomtemperaturefor30-60minutes,protectedfromlight.
Note:Fora384-wellplate,add7.5µLEnhancer.
7.5MonitorthefluorescenceincreasewithafluorescenceplatereaderatEx/Em=420/480nm.
| References&Citations |  PrinterFriendlyVersion |
1. QiX,NgKT,LianQZ,LiuXB,LiCX,GengW,LingCC,MaYY,YeungWH,TuWW,FanST,LoCM,ManK.(2014)Clinicalsignificanceandtherapeuticvalueofglutathioneperoxidase3(GPx3)inhepatocellularcarcinoma.Oncotarget.
2. SongJ,YuY,XingR,GuoX,LiuD,WeiJ,SongH.(2014)Unglycosylatedrecombinanthumanglutathioneperoxidase3mutantfromEscherichiacoliisactiveasamonomer.SciRep,4,6698.
3. BrutschSH,WangCC,LiL,StenderH,NezirogluN,RichterC,KuhnH,BorchertA.(2014)Expressionofinactiveglutathioneperoxidase4leadstoembryoniclethalityandinactivationoftheAlox15genedoesnotrescuesuchknock-inmice.AntioxidRedoxSignal.
4. CorreaLB,ZanettiMA,DelClaroGR,dePaivaFA,daLuzESS,NettoAS.(2014)Effectsofsupplementationwithtwosourcesandtwolevelsofcopperonmeatlipidoxidation,meatcolourandsuperoxidedismutaseandglutathioneperoxidaseenzymeactivitiesinNellorebeefcattle.BrJNutr,112,1266.
5. KoretsiV,KirschneckC,ProffP,RomerP.(2014)Expressionofglutathioneperoxidase1inthespheno-occipitalsynchondrosisanditsroleinROS-inducedapoptosis.EurJOrthod.
6. BerminghamEN,HeskethJE,SinclairBR,KoolaardJP,RoyNC.(2014)Selenium-EnrichedFoodsAreMoreEffectiveatIncreasingGlutathionePeroxidase(GPx)ActivityComparedwithSelenomethionine:AMeta-Analysis.Nutrients,6,4002.
7. TrevisanR,MelloDF,Uliano-SilvaM,DelapedraG,ArlM,DafreAL.(2014)Thebiologicalimportanceofglutathioneperoxidaseandperoxiredoxinbackupsystemsinbivalvesduringperoxideexposure.MarEnvironRes,101C,81.
8. SakamotoT,MaebayashiK,NakagawaY,ImaiH.(2014)DeletionofthefourphospholipidhydroperoxideglutathioneperoxidasegenesacceleratesaginginCaenorhaBDitiselegans.GenesCells,19,778.
9. PressDJ,McNeilNM,HambrookM,BackTG.(2014)EffectsofMethoxySubstituentsontheGlutathionePeroxidase-likeActivityofCyclicSeleninateEsters.JOrgChem,79,9394.
10. RuszkiewiczJ,AlbrechtJ.(2014)ChangesoftheThioredoxinSystem,GlutathionePeroxidaseActivityandTotalAntioxidantCapacityinRatBrainCortexDuringAcuteLiverFailure:ModulationbyL-histidine.NeuRochemRes.
