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AntibodiesOnline/Glutathione Fluorescent Detection Kit/ABIN577647/5 x 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 Fluorescent Detection Kit/ABIN577647/5 x 96 tests
Antigen
Glutathione
Reactivity
Chemical
22Chemical
1AminoAcid
1Chicken
1Cow(Bovine)
1Dog(Canine)
1Goat
1GuineaPig
1Human
1Monkey
1Mouse(Murine)
1Pig(Porcine)
1Rabbit
1Sheep(Ovine)
MinimumDetectionLimit
42nM
Application
BiochemicalAssay(BCA)
Options
Bulkdiscount
Supplier
SupplierProductNo.
PurposeTheDetectX®Glutathionekitisdesignedtoquantitativelymeasureglutathione(GSH),andoxidizedglutathione(GSSG)presentinavarietyofsamples.
BrandDetectX®
SampleTypeBlood,Serum,Plasma,ErythrocyteLysates,Urine,CellLysate,TissueSamples
DetectionMethodFluorometric
SpecificitySampleTypesvalidated:WholeBlood,Serum,Plasma,Erythrocytes,Urine,CellLysatesandTissueSamples
Sensitivity45nMintheFreeGSHand48nMintheTotalGSHassays.
CharacteristicsTheGlutathione(GSH)Fluorometrickitutilizesaproprietarynon-fluorescentmolecule,ThioStar®,thatcovalentlybindstothefreethiolgrouponGSHtoyieldahighlyfluorescentproduct.AftermixingthesampleorstandardwithThioStar®andincubatingatroomtemperaturefor15minutes,theGSH-generatedsignalisreadat510nminafluorescentplatereaderwithexcitationat390nm.AdditionofareactionmixtureconvertsalltheGSSGintofreeGSH,whichthenreactswiththeexcessThioStar®duringasecond15minuteincubation,yieldingthesignalrelatedtoTotalGSHcontent.Glutathione(GSH)isthehighestconcentrationnon-proteinthiolinmammaliancellsandispresentinconcentrationsof0.5to10mM.GSHplaysakeyroleinmanyBIOLOGicalprocesses,includingthesynthesisofproteinsandDNA,thetransportofaminoacids,andtheprotectionofcellsagainstoxidation.Harmfulhydrogenperoxidecellularlevelsareminimizedbytheenzymeglutathioneperoxidase(GP)usingGSHasareductant.TheoxidizedGSHdimer,GSSG,isformedfromGSHandperoxidebytheGPreaction.AnimportantroleofGSSGintheNFkBactivatingsignalcascadeissuggestedbythefactthatthepotentNFkBinducerTPAincreasesintracellularGSSGlevelsandGSSG/GSHratios.
ComponentsBlack96WellPlate1or5each
GlutathioneStandardGlutathioneat250μMinaspecialstABIlizingsolution.100μLor3μL
ThioStar®DetectionreagentThioStarthioldetectionsubstratestoredinaziplocpouchwithdesiccant.ReconstitutewithdryDMSO.2Plasticvialsor4Glassvials
DryDMSODryDimethylsulfoxidesolventovermolecularsieves.Maybestoredatroomtemperature.4mLor20mL
AssayBufferConcentrateA2Xbufferconcentratecontainingdetergentsandstabilizersthatmustbedilutedwithdeionizedordistilledwater.35mLor200mL
NADHPConcentrateReducedß-nicotinamideadeninedinucleotide2-phosphate(NADPH)asastablesolution.300μLor1.4mL
GlutathionereductaseConcentrateGlutathioneReductase(GR)asastablesolution.300μLor1.4mL
OxidizedGlutathioneControlOxidizedGlutathione(GSSG)inaspecialstabilizingsolution.ThisisanoptionalcontrolsolutiontoensureNADPH/GRperformance.300μL
MaterialnotincludedDistilledordeionizedwaterRepeaterpipetwithdisposabletipscapableofdispensing25μL.
Aqueous5-sulfo-salicylicaciddihydrate(SSA)solutionat5%weight/volume(1gofSSAper20mLofwater)fortreatingsamplestoremoveprotein.
WerecommendSigma-Aldrich.
Fluorescence96wellplatereadercapableofreADIngfluorescentemissionat510nm,withexcitationat390nm.
Pleasecontactyourplatereadermanufacturerforsuitablefiltersets.
Setplateparametersfora96-wellCorningCostar3650plate.
See:www.arborassays.com/resources/#general-infoforplatedimensiondata.
Thesensitivityoffluorescentassaysisdependantonthecapabilitiesoftheplatereader.
Ifyourplatereaderhasadjustablegainyoucanmodifythesignalsobtainedfromtheassaybyincreasingordecreasingthegainsettings,bychangingtheaperturesettingsformonochromatorbasedreaders,orbychangingthebandpasswidthoftheemissionand/orexcitationfiltersonsomereaders.
Pleasereviewtheplatereadermanualfordetails.signalsexpressedbyplatereadersarerelativeFluorescentunits(rFu)andthevaluesgivenintheinsertwereobtainedonourplatereaders.therFunumbersyouobtainmaybedifferentfromthese,buttheassayresultsshouldbesimilar.
Softwareforconvertingrawrelativefluorescentunit(FLU)readingsfromtheplatereaderandcarryingoutfourparameterlogisticcurve(4PLC)fitting.
BackgroundGlutathione(L-γ-glutamyl-L-cysteinylglycine,GSH)isthehighestconcentrationnon-proteinthiolinmammaliancellsandispresentinconcentrationsof0.5-10mM1.GSHplaysakeyroleinmanybiologicalprocesses,includingthesynthesisofproteinsandDNA,thetransportofaminoacids,andtheprotectionofcellsagainstoxidation.Harmfulhydrogenperoxidecellularlevelsareminimizedbytheenzymeglutathioneperoxidase(GP)usingGSHasareductant2.Shnhoo2honhonohhoTheoxidizedGSHdimer,GSSG,isformedfromGSHandperoxidebytheGPreaction(seebelow).AnimportantroleofGSSGintheNFΚBactivatingsignalcascadeissuggestedbythefactsthatthepotentNFΚBinducer,tetradecanoylphorbolacetate,increasesintracellularGSSGlevelsandGSSG/GSHratios3.h2oh2o2GlutathionePeroxidaseGSSGGShGlutathionereductaseGlutathioneS-transferasenADPhnADP+GShConjugateGlutathioneS-transferases(GST)areanimportantgroupofenzymesthatcatalyzethenucleophilicadditionofGSHtoelectrophiles.Theyareencodedby5genefamilies,4encodecytosolicGSTandoneencodesthemicrosomalformofGST.Theyhavebeenimplicatedinanumberofdiseases.InasthmaarachidonicacidisconvertedtounstableleukotrieneA(LTA).LTAiseitherhydrated444toformLTBoritisconjugatedtoGSHbyaGST,leukotrieneCsynthase,toformleukotrieneC.444LTCanditsderivativeLTDareimportantmoleculesinbronchialasthma.LeukotrieneCsynthase444isthereforeanimportanttherapeutictarget.IthasalsobeenshownthatincreasedexpressionofGSTscanleadtodrugresistance.Threeglutathioneadductsofthedrugmelphalan,usedtotreatovariancancerandmultiplemyeloma,havebeenisolatedfromreactionsinvolvinghumanmicrosomalGSTs
ResearchAreaMetabolism,AminoAcids,Enzymes
ApplicationNotesGSHisidenticalacrossspeciesandweexpectthiskitmaymeasureGSHfromsourcesotherthanhuman.
TheendusershouldevaluaterecoveriesofGSHinsamplesfromotherspeciesbeingtested.
Ifsamplesneedtobestoredaftercollection,werecommendstoringthemat-70 °Corlower,preferablyafterbeingfrozeninliquidnitrogen.
Thisassayhasbeenvalidatedforhumanwholeblood,serum,EDTAandheparinplasma,urine,andisolatederythrocytes.
MostcelllysatesandtissuehomogenatesshouldalsobecompatIBLe.
Samplescontainingvisibleparticulateshouldbecentrifugedpriortousing.
Allsampleswillbedeproteinizedwith5 %SSA(seepage6forpreparation),pleaseseesamplespecificinformationbelowfordetails.
ThistreatmentremovesanyproteinthiolspresentinthesamplesandalsoslowsoxidationoffreeGSH.
Plate96wells
ProtocolThekitisuniqueinthatbothfreeandoxidizedglutathionearedetectedinthesamewellinthemicrotiterplate.
Noseparationorwashingisrequired.
TotalglutathioneisthesumofGSSGplusGSH.
Pleasereadthecompletekitinsertbeforeperformingthisassay.
AGSHstandardisprovidedtogenerateastandardcurvefortheassayandallsamplesshouldbereadoffthestandardcurve.
Thekitutilizesaproprietarynon-fluorescentmolecule,ThioStar®,thatwillcovalentlybindtothefreethiolgrouponGSHtoyieldahighlyfluorescentproduct.
AftermixingthesampleorstandardwithThioStar®andincubatingatroomtemperaturefor15 minutes,thefluorescentproductisreadat510nminafluorescentplatereaderwithexcitationat390nm.
TheconcentrationoftheGSHinthesampleiscalculated,aftermakingasuitablecorrectionforanydilutionofthesample,usingsoftwareavailablewithmostfluorescenceplatereaders.
Freeglutathione,GSH,isreadfirstafter15 minutes,followedbyadditionofareactionmixturethatconvertsalltheoxidizedglutathione,GSSG,intofreeGSH,whichthenreactswiththeexcessThioStar®toyieldthesignalrelatedtoTotalGSHcontent.
ThetotalconcentrationofGSHgeneratedinthesampleiscalculatedfromthegeneratedsignal.
Wehaveprovideda96wellplateformeasurementbutthisassayisadaptableforhigherdensityplateformats.
TheendusershouldensurethattheirHTSblackplateissuitableforusewiththesereagentspriortorunningsamples.
ReagentPreparation

Allowthekitreagentstocometoroomtemperaturefor30minutes.
Ensurethatallsampleshavereachedroomtemperatureandhavebeendilutedasappropriatepriortorunningtheminthekit.assayBufferPreparetheAssayBufferbydilutingthesuppliedAssayBufferConcentratewithanequalvolumeofdeionizedwater.
Mixthoroughly.
Stableat4°Cfor3months.
SampleDiluentPreparetheSampleDiluentbydilutingonepart5%SSA1:5withfourpartsdilutedAssayBufferandvortexthoroughly.
ThepHoftheSampleDiluentmustbe>6.
SampleDiluentcanbestoredat4°Cforonemonth.
StandardPreparationGSHStandardsarepreparedbylabelingeighttesttubesas#1through#8.
Brieflyvortextomixandthenspinthevialofstandardinamicrocentrifugetoensurecontentsareatbottomofvial.
Pipet450μLofSampleDiluentintotube#1and250μLintotubes#2to#8.
Carefullyadd50μLoftheGlutathioneStandardtotube#1andvortexcompletely.
Take250μLoftheGSHsolutionintube#1andaddittotube#2andvortexcompletely.
Repeatthisfortubes#3through#8.
TheconcentrationofGSHintubes1through8willbe25,12.5,6.25,3.125,1.56,0.781,0.391and0.195μM.
UseallStandardswithin1hourofpreparation.
Std1Std2Std3Std4Std5Std6Std7Std8SampleDiluentVol(μL)450250250250250250250250additionStockStd1Std2Std3Std4Std5Std6Std7Volofaddition(μL)50250250250250250250250FinalConc(μM)2512.56.253.1251.560.7810.3910.195®www.ArborAssays.com9ControlPreparation(optional)ThisoptionalcontrolsolutionforensuringcompleteconversionofGSSGtoGSHcanbepreparedbyadding5μLofOxidizedGlutathioneControlto245μLofSampleDiluent.usewithin2hours.
TheControlPreparationensuresthattheNADPHandGlutathioneReductasesystempreparedbelowintheReactionMixturesectionwilladequatelyreduceGSSGtoGSH.
IfthisoptionalcontrolisrunitshouldyieldavalueforTotalGlutathioneofapproximately10±2μM.thioStar®DetectionreagentAllowtheziplocbagtowarmcompletelytoroomtemperaturepriortoopeningandremovethevialofThioStarReagent.
AddthevolumeofDMSOprovidedtothevialaccordingtothetablebelow.
Vortexthoroughly.
StoreanyunusedreconstitutedDetectionReagentat4°Cintheziplocpouchwithdesiccantandusewithin2months.
KitK006-F1KitK006-F5VialPartNumberC021-1EA,PlasticvialC036-1EA,GlassvialVolumeofDMSOtoaddpervial1.5mL3.5mLFor#ofWellsUpto60Upto140reactionMixturePreparetheReactionMixturebyvortexingthevialsofGlutathioneReductaseandNADPHConcentratesandthendilutingoneparteachNADPHandGlutathioneReductaseConcentrates1:10intoeightpartsAssayBuffer.
Vortexthoroughly.
SeeTableforsuitablevolumes.
StoreanyunusedReactionMixtureat4°Cinanambervialfornomorethan2days.reactionMixDilutiontable1/2PlateonePlateNADPHConcentrate150μL275μLGlutathioneReductaseConcentrate150μL275μLAssayBuffer1.2mL2.2mL®10EXPECTASSAYARTISTRYASSAyProtoCol-FreeAnDtotAlGShWerecommendthatallstandardsandsamplesberuninduplicatetoallowtheendusertoaccuratelydetermineGShconcentrations.1.
Usetheplatelayoutsheetonthebackpagetoaidinpropersampleandstandardidentification.
Setplateparametersfora96-wellCorningCostar3650plate.2.
Pipet50μLoftreatedsamples,standardsorcontrolintowellsintheplate.3.
Pipet50μLofSampleDiluentintoZerowellsintheplate.4.
Add25μLoftheThioStarReagenttoeachwellusingarepeaterpipet.5.
Gentlytapthesidesoftheplatetoensureadequatemixingofthereagents.6.
Incubateatroomtemperaturefor15minutes.7.
Readthefluorescentsignalfromeachwellinaplatereadercapableofreadingthefluorescentemissionat510nmwithexcitationat370-410nm.
ThisdatawillbeusedtodetermineFreeGSHconcentration.8.
Add25μLoftheReactionMixturetoeachofthewellsusingarepeaterpipet.9.
Gentlytapthesidesoftheplatetoensureadequatemixingofthereagents.10.
Incubateatroomtemperaturefor15minutes.11.
Readthefluorescentemissionat510nmwithexcitationat370-410nm.
ThisdatawillbeusedtodetermineTotalGSHconcentration.totalGShContentonlyTotalGSHcontentcanbedetermineddirectlybyleavingoutsteps5,6and7.

SamplePreparation

AllsamplesmustbetreatedwiththeSSAsolutionpreparedonpage6.AlloftheSSAtreatedcentrifugedsupernatantsmusthavetheirSSAconcentrationbroughtdownto1%SSAbydilutionwithAssayBuffer.Furtherdilutionsofthesample,usingSampleDiluent(seepage9forpreparation),maybenecessarytoallowtheGSHconcentrationtobemeasurementintheassay.Detailedinstructionsfollow.AllsamplesandstandardsmustbeinSampleDiluentbeforestartingtheassay.useallsampleswithin2hoursofdilution.WholeBlood,Serum,eDtAorheparinPlasma,orurineThoroughlymixsamplewithanequalvolumeofcold5%SSA.Incubatefor10minutesat4°C.Centrifugeat14,000rpmfor10minutesat4°C.Collectthesupernatant.Ifthesupernatentcontainsparticulates,re-centrifugethesupernatantfor15minutesandcollecttheclarifiedsecondsupernatant.Samplescanbestoredinaliquotsat≤-70°Coranalyzedimmediately.AtthispointtheSSAconcentrationwillbe2.5%.Thesupernatantmustbediluted1:2.5withAssayBufferbymixingonepartwith1.5partsofAssayBuffer.TheSSAconcentrationwillbe1%.Thesamplewillhavebeendiluted1:5atthispoint.AllfinaldilutionsaretobemadeinSampleDiluent.TreatedWholeBloodmustbefurtherdilutedatleast1:20forarecommendedfinaldilutionof≥1:100.ForTreatedPlasmaandTreatedUrineafinaldilutionof≥1:5isrecommended,butfurtherdilutionsinSampleDiluentmaybenecessary.

CalculationofResults

AveragetheduplicateFLUreadingsforeachstandardandsample.
Createastandardcurvebyreducingthedatausingthe4PLCfittingroutineontheplatereader,aftersubtractingthemeanFLUsforthezerostandard.
Thesampleconcentrationsobtainedshouldbemultipliedbythedilutionfactortoobtainneatsamplevalues.

RestrictionsForResearchUseonly
PrecautionofUseAswithallsuchproducts,thiskitshouldonlybeusedbyqualifiedpersonnelwhohavehadlaboratorysafetyinstruction.
Thecompleteinsertshouldbereadandunderstoodbeforeattemptingtousetheproduct.
Sulfosalicylicacidisastrongacidsolutionandshouldbetreatedlikeanyotherlaboratoryacid.
Dimethylsulfoxideisapowerfulaproticorganicsolventthathasbeenshowntoenhancetherateofskinabsorptionofskin-permeablesubstances.
Wearprotectivegloveswhenusingthesolventespeciallywhenitcontainsdissolvedchemicals.
NOTE:DMSOcandissolvecertainplasticsusedintroughsusedforholdingsolutionsformultichannelpipets.thiostar®thioldetectionreagentshouldbestoredat4°cinthedesiccatedpouch.allowdesiccatedpouchtowarmtoroomtemperaturepriortoopening.thiostarwillreactwithstrongnucleophiles.
Bufferscontainingthepreservativessodiumazide,Proclin™andKathon™willreactwiththesubstrate.
ReconstitutedThioStarinDMSOstoredat4°Cinthedesiccatedpouch.
Itcanbeusedupto2monthslater.
ThebackgroundonthereconstitutedThioStarwillincreaseslowlyovertimebuttheincreasewillnotaffecttheassayresultsobtained.
Storage4°C
StorageCommentAllcomponentsofthiskitshouldbestoredat4°Cuntiltheexpirationdateofthekit.DMSO,whenstoredat4°C,willfreeze.Canbestoredtightlycappedatroomtemperature.
SupplierImages
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