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AntibodiesOnline/Glutathione Reductase Fluorescent Kit/ABIN577651/96 tests188bio精品生物—专注于实验室精品爆款的电商平台 - 蚂蚁淘旗下精选188款生物医学科研用品
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AntibodiesOnline/Glutathione Reductase Fluorescent Kit/ABIN577651/96 tests
Antigen
GlutathioneReductase(GSR)
  • GR
  • DDBDRAFT_0168952
  • DDBDRAFT_0231410
  • DDB_0168952
  • DDB_0231410
  • 2151
  • CG2151
  • DTR
  • Dm-TrxR
  • DmTR
  • DmTrx
  • DmTrxR
  • DmTrxR-1
  • DmelCG2151
  • Gr
  • Trx
  • TrxR
  • TrxR-1
  • Trxr1
  • anon-WO03040301.185
  • anon-WO03040301.187
  • dTrxR
  • dmtrxr-1
  • gr
  • l(1)G0154
  • l(1)G0379
  • l(1)G0477
  • l(1)G0481
  • trxr-1
  • ATGR2
  • EMB2360
  • glutathionereductase
  • AI325518
  • D8Ertd238e
  • Gr-1
  • Gr1
  • gor1
  • GSR
  • glutathionereductase,gro-2
  • glutathionereductase
  • GR;GRase
  • Glutathionereductase(GR)(GRase)
  • Thioredoxinreductase-1
  • glutathionereductase1
  • guttershapedroot
  • GR
  • gor
  • GSR
  • PTRCGR1
  • GSR1
  • Gsr_2
  • GSHR1
  • gsr
  • LbGR
  • pgr1
  • GSHR2
  • GLR2
  • Bcen_2392
  • RPE_1989
  • HCAG_02219
  • SJAG_01162
  • Trxr-1
  • PF14_0192
  • PtrcGR2
  • Gsr
  • gsr1
  • Gusr
Alternatives
Sheep(Ovine)GlutathioneReductaseELISAKit
Showmore
1Sheep(Ovine)GlutathioneReductaseELISAKit
10Rat(Rattus)GlutathioneReductaseELISAKit
2RabbitGlutathioneReductaseELISAKit
2Pig(Porcine)GlutathioneReductaseELISAKit
13Mouse(Murine)GlutathioneReductaseELISAKit
2MonkeyGlutathioneReductaseELISAKit
13HumanGlutathioneReductaseELISAKit
1Horse(Equine)GlutathioneReductaseELISAKit
2GuineaPigGlutathioneReductaseELISAKit
1GoatGlutathioneReductaseELISAKit
1Dog(Canine)GlutathioneReductaseELISAKit
1Cow(Bovine)GlutathioneReductaseELISAKit
1ChickenGlutathioneReductaseELISAKit
MinimumDetectionLimit
9µU/mL
Application
BiochemicalAssay(BCA)
Options
Bulkdiscount
Supplier
SupplierProductNo.
PurposeTheDetectX®GlutathioneReductase(GR)FluorescentActivitykitisdesignedtoquantitativelymeasureglutathionereductase(GR)activityinavarietyofsamples.
BrandDetectX®
SampleTypeSerum,Plasma,RedBloodCells,CellLysate
DetectionMethodFluorometric
SpecificitySampleTypesvalidated:Serum,Plasma,RBCsandCellLysates
Sensitivity0.009mU/mL
CharacteristicsTheGlutathioneReductase(GR)FluorescentActivityKitdeterminesGRactivitybytheamountofGSHgeneratedfromthereductionofGSSGwithanon-fluorescentmolecule,ThioStar®,thatcovalentlybindsthefreethiolgrouponGSHtoyieldahighlyfluorescentproduct.ThemostwidelyusedproceduretomeasureGRistomonitortheoxidationofNADPHasadecreaseinabsorbanceat340nm.HowevermanyBIOLOGicalmoleculesabsorblightat340nm,plusthedetectionsystemgivesverylowODreADIngs.TheARBORASSAYSGlutathioneReductaseFluorescentActivitykitovercomestheseproblemsofmeasuringGRactivitybythedirectdetectionoftheGSHformedfromoxidizedglutathione.Glutathionereductase(GR)playsanindirectbutessentialroleinthepreventionofoxidativedamagewithinthecellbyhelpingtomaintainappropriatelevelsofintracellularglutathione(GSH).GSH,inconjuctionwiththeenzymeglutathioneperoxidase(GP),istheactingreductantresponsIBLeforminimizingharmfulhydrogenperoxidecellularlevels.TheregenerationofGSHiscatalyzedbyGR.GRisanubiquitous100-120kDadimericflavoproteinthatcatalyzesthereductionofoxidizedglutathione(GSSG)toreducedglutathione,usingß-nicotinamidedinucleotidephosphate(NADPH)asthehydrogendonor.MoleculessuchasNADPHactashydridedonorsinavarietyofenzymaticprocesses.NADPHhasbeensuggestedtoalsoactasanindirectlyoperatingantioxidant,givenitsroleinthere-reductionofGSSGtoGSHandthusmaintainingtheantioxidativepowerofglutathione.
ComponentsBlackHalfArea96WellPlate
GlutathioneReductaseStandard40μLGlutathioneReductaseat200mU/mLinaspecialstABIlizingsolution.
ThioStar®DetectionReagent1vialThioStarthioldetectionsubstratestoredinadesiccator.ReconstitutewithdryDMSO.
DryDMSO2mLDryDimethylsulfoxidesolventovermolecularsieves.Maybestoredatroomtemperature.
AssayBufferConcentrate60mLA2xconcentratedphosphatebuffercontainingdetergentsandstabilizers.
NADPH1vialReducedß-nicotinamideadeninedinucleotide2-phosphatefreezedriedwithstabilizersstoredinadesiccator.
NADPHDiluent5mLAphosphatebuffercontainingdetergentsandstabilizers.
OxidizedGlutathione3mLOxidizedGlutathione(GSSG)inaspecialstabilizingsolution.
MaterialnotincludedRepeaterpipetwithdisposabletipscapableofdispensing15μLand25μL.
Fluorescence96wellplatereadercapableofreadingfluorescentemissionat510nm,withexcita-tionat390nm.
Softwareforconvertingrawrelativefluorescentunit(FLU)readingsfromtheplatereaderandcarryingoutfourparameterlogisticcurve(4PLC)fitting.Setplateparametersfora96-wellCorningCostar3686plate.
See:http://www.Ar-borAssays.com/resources/lit.aspforplatedimensiondata.
TheSupplierhasavailableforfreedownloadonourwebsiteanExcelspreadsheetusefulinsub-tractingoutsamplethiolbackgroundat:http://www.arborassays.com/resources/lit.asp
AlternativeNameGlutathioneReductase(GSRELISAKitAbstract)
BackgroundGlutathionereductase(GR)playsanindirectbutessentialroleinthepreventionofoxidativedam-agewithinthecellbyhelpingtomaintainappropriatelevelsofintracellularglutathione(GSH).GSH,inconjuctionwiththeenzymeglutathioneperoxidase(GP),istheactingreductantrespon-sibleforminimizingharmfulhydrogenperoxidecellularlevels1.TheregenerationofGSHiscata-lyzedbyGR2.GRisanubiquitous100-120kDadimericflavoproteinthatcatalyzesthereductionofoxidizedglutathione(GSSG)toreducedglutathione,usingß-nicotinamidedinucleotidephos-phate(NADPH)asthehydrogendonor3.MoleculessuchasNADPHactashydridedonorsinavarietyofenzymaticprocesses.NADPHhasbeensuggestedtoalsoactasanindirectlyoperatingantioxidant,givenitsroleinthere-reductionofGSSGtoGSHandthusmaintainingtheantioxida-tivepowerofglutathione.ThegeneralGRreactionisshownbelow:GlutathioneReductaseGSSG2GSHNADPH+H+NADP+ThemostwidelyusedproceduretomeasureGRistomonitortheoxidationofNADPHasade-creaseinabsorbanceat340nm4.Howeverthismethodsuffersfromtheabsorbanceofmanybiologicalmoleculesat340nm.ThisDetectX®assaydeterminesGRactivitybydirectlymeasuringtheamountofGSHgeneratedfromthereductionofGSSGbyreactingtheGSHwithanon-fluorescentmolecule,ThioStar®,tocovalentlybindthefreethiolgrouponGSHandyieldahighlyfluorescentproduct
ResearchAreaMetabolism,Enzymes
ApplicationNotesThisassayhasbeenvalidatedforhumanserum,EDTAandheparinplasma,andisolatederythro-cytes.
Mostcelllysatesshouldalsobecompatible.
Samplescontainingvisibleparticulateshouldbecentrifugedpriortousing.
GRactivityvariesacrosstissuesandspecies,howeverweexpectthiskittomeasureGRactivityfromsourcesotherthanhuman.
Theendusershouldevaluatere-coveriesofGRactivityinsamplesfromotherspeciesbeingtested.
AssayTime1h
Plate96wells
ProtocolAGRstandardisprovidedtogenerateastandardcurvefortheassayandallsamplesshouldbereadoffthestandardcurve.
Thekitutilizesaproprietarynon-fluorescentmolecule,ThioStar®,thatwillcovalentlybindtothefreethiolgrouponGSHgen-eratedinthereductionofoxidizedglutathione(GSSG)toyieldahighlyfluorescentproduct.
AftermixingthesampleorstandardwithThioStar®andincubatingatroomtemperature,thefluorescentproductisreadat510nminafluorescentplatereaderwithexcitationat390nm.
Backgroundthiolcontentisreadfirstafter5minutes,followedbyadditionofGSSGandNADPHwhichusesthestandardorsampleGRtoconverttheoxidizedglutathione,GSSG,intofreeGSH,whichthenreactswiththeThioStar®toyieldthesignalrelatedtoGRactivityTheactivityofGRinthesampleiscalculatedfromthegeneratedsignal.
Wehaveprovideda96wellplateformeasure-mentbutthisassayisadaptableforhigherdensityplateformats.
TheendusershouldensurethattheirHTSblackplateissuitableforusewiththesereagentspriortorunningsamples.
ReagentPreparation

Allowthekitreagentstocometoroomtemperaturefor30minutes.
Werecommendthatallstan-dardsandsamplesberuninduplicatetoallowtheendusertoaccuratelydetermineGRactivity.
Ensurethatallsampleshavereachedroomtemperatureandhavebeendilutedasappropriatepriortorunningtheminthekit.
AssayBufferPreparationPreparetheAssayBufferbydilutingonepartofthe2xAssayBufferConcentrate1:2withonepartdeionizedwater.
Itisstableforupto3monthswhenstoredat4°C.
StandardPreparationGRStandardsarepreparedbylabelingsixtesttubesas#1through#6.
Brieflyspinvialofstan-dardinamicrocentrifugetoensurecontentsareatbottomofvial.
Pipet390μLofAssayBufferintotube#1and200μLintotubes#2to#6.
Carefullyadd10μLoftheGlutathioneReductaseStandardtotube#1andvortexcompletely.
Take200μLoftheGRsolutionintube#1andaddittotube#2andvortexcompletely.
Repeattheseserialdilutionsfortubes#3through#6.
TheconcentrationofGRintubes1through6willbe5,2.5,1.25,0.625,0.3125,and0.156mU/mL.
UseallStandardswithin2hourofpreparation.
Std1Std2Std3Std4Std5Std6BufferVolume(μL)390200200200200200AdditionStockStd1Std2Std3Std4Std5VolumeofAddition(μL)10200200200200200FinalConc(mU/mL)52.51.250.6250.31250.156ThioStar®DetectionReagentRemovethevialofThioStar®Reagentfromthedesiccatorandadd1.8mLoftheprovidedDMSOtothevial.
Vortexthoroughly.
StoreanyunusedreconstitutedDetectionReagentat4°Cinthedesiccatorandusewithin2months.
NADPHAdd3mLoftheNADPHDiluenttotheNADPHvialandvortexthoroughly.
Storeanyunusedre-constitutedNADPHat4°Cfornomorethan2weeks.

SamplePreparation

AnysamplesrequiringlargerdilutionsorwithGRactivitiesoutsidethestandardcurverangeshouldbedilutedfurtherwithAssayBuffertoobtainreadingswithinthestandardcurve.Serumand

AssayProcedure
  1. Usetheplatelayoutsheetonthebackpageoftheinserttoaidinpropersampleandstandardidentification.Setplateparametersfora96-wellCorningCostar3686plate.
    2.Pipet25μLofsamplesorstandardsintoduplicatewellsintheplate.
    3.Pipet25μLofAssayBufferintoduplicatewellsastheZerostandard.
    4.Add15μLoftheThioStar®DetectionReagenttoeachwellusingarepeaterpipet.
    5.Gentlytapthesidesoftheplatetoensureadequatemixingofthereagents.
    6.Incubateatroomtemperaturefor5 minutes.
    7.Readthefluorescentsignalfromeachwellinaplatereadercapableofreadingthefluorescentemissionat510nmwithexcitationat370-410nm.Pleasecontactyourplatereadermanufacturerforsuitablefiltersets.Thisdatawillbeusedtosubtractanybackgroundthiolsignalinsamples.
    8.Add25μLoftheOxidizedGlutathionetoeachofthewellsusingarepeaterpipet.
    9.Add25μLoftheNADPHtoeachofthewellsusingarepeaterpipet.10.Gentlytapthesidesoftheplatetoensureadequatemixingofthereagents.11.Incubateatroomtemperaturefor15 minutes.12.Readthefluorescentemissionat510nmwithexcitationat370-410nm.Pleasecontactyourplatereadermanufacturerforsuitablefilters.
CalculationofResults

TheSupplierhasavailableforfreedownloadonourwebsiteanExcelspreadsheetusefulinsub-tractingoutsamplethiolbackgroundat:http://www.arborassays.com/resources/lit.aspAftersubtractingthebackgroundthiolFLUreadingsforeachwellfromstep6,averagethedupli-cateFLUreadingsforeachstandardandsample.
Createastandardcurvebyreducingthedatausingthe4PLCfittingroutineontheplatereader,aftersubtractingthemeanFLUsforthezerostandard.
Theactivitiesobtainedshouldbemultipliedbythedilutionfactortoobtainneatsamplevalues.
Orusetheonlinetoolfromhttp://www.myassays.com/arbor-assays-glutathione-reductase-fluorescent-kit.assaytocalculatethedata.*

RestrictionsForResearchUseonly
PrecautionofUseAswithallsuchproducts,thiskitshouldonlybeusedbyqualifiedpersonnelwhohavehadlabo-ratorysafetyinstruction.
Thecompleteinsertshouldbereadandunderstoodbeforeattemptingtousetheproduct.
Dimethylsulfoxideisapowerfulaproticorganicsolventthathasbeenshowntoenhancetherateofskinabsorptionofskin-permeablesubstances.
Wearprotectivegloveswhenusingthesolventespeciallywhenitcontainsdissolvedchemicals.
ReconstitutedThioStar®DetectionReagentshouldbestoredat4°Cinthedesic

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