CA-G056 | |
Name | EliteTM Glutathione GSH/GSSGRatioAssayKit(Fluorescence) |
Description | TheElite™GlutathioneGSH/GSSGRatioAssayKitprovidesanultrasensitiveassaytoquantifyGSHinthesample.Thiskitusesaproprietarynon-fluorescentdyethatbecomesstronglyfluorescentuponreactingwithGSH.Withaone-stepfluorimetricmethod,thekitcandetectaslittleas1picomoleofGSHorGSSGina100μlassayvolume. |
Application | Theassaycanbeconvenientlyperformedina96-wellor384-wellmicrotiter-plateformatandreADIlyadaptedtoautomationwithoutaseparationstep.ItssignalcanbeeasilyreadbyafluorescencemicroplatereaderatEx/Em=490/520nm. |
Size | 200assays |
Detection | Fluorescencemicroplatereader |
Clickherefor DATASHEET | Clickherefor MSDS |
FrequentlyAskedQuestionsandAnswers:
1.Q:Whatwouldyousuggesttopreparethecellsamplesforthisassay?
Answer:Allthedetergentsinvariouslysisbufferswillcausebackgroundnoisefortheassay.Thetraditionalwaytodoitisthe"freezeandthaw"method(i.e.freezefor20-30minutesat-80Cthenthawatroomtemperature,2-3times),butitistootedious.Wefoundthat0.2%SDSgivesthebestresultsamongallthedetergentstested.
2.Q:DoesthisKitworkforplantsamples?Couldyourecommendaspecialprotocoltomeasuregluthationeinplants?
Answer:Aslongasthesampl chop2gofplantparts(leaf,root,grain,etc.)andgrounditinahomogenizerfor2minwith5-10mlof0.1NHCl;centrifugetopbtainthe GSHcontainingsupernatant,thenadjustpHto6.5fortheGSHassay.
3.Q:Mysampleisfrozenbraintissue,howshouldIprepareitforthesaasy?
Answer:ItisbetterusePBS,andsimplyhomogenizeit,thenusethesupernatant.
4.Q:DoesthiskitmeasureGSSGandGSHdirectly?
Answer:Thiskitcanbeusedformeasuringtotalandthereducedform(GSH)directly,theoxidizedform(GSSG)iscalculated[thetotalminusGSH].TheGSSGprobeisareducingagentthatreducesGSSGtoGSH(inordertomeasuretotalGSH).Thedetectionlimitisaround0.1~0.3uM.
5.Q:Iworkwithhumanprimarycells.HowmanycellsshouldIuseintheassay?
Answer:Suggesttostartwith10,000cells/wellfirst.
6.Q:Isanyofthecomponentsinthiskitfromanimals?
Answer:No.Theonlyenzyme(inComponentE)isfromBaker"sYeast(S.cerevisiae).
7.Q:Forthefluorescentdetection,anemissionfilterwith520nmisrecommended.Butwehaveonly535nmfilter.Woulditworkforthismeasurement?
Answer:Yes.
8.Q:Wedon"thavethefiltersfor490/520(nm).Whatwehavearefor395/535(nm).Woulditwork?
Answer:No.theemissionisOK,buttheexcitationisnot.Thelowestexcitationwavelengthisat460nm.
9.Q:I"vetriedthiskit,butitdidn"twellformysamples.HereistheprotocolIusedforobtainingcellculturelysates.Couldyoutakealookandgiveyoursuggestion?
Answer:
-trypsinizefor2-6min,stoptrypsination;[WesuggesttoavoidtrypsinandtryusingarubberscrapperorEDTAtoharvestcells,thencentrifugethecellsandusethesupernatant]
-centrifugepelletdownat180g,5min,4°C,thenwashwith5mlcoldPBS;countcells;[Fine]
-centrifugepelletdownat180g,5min,4°C;[Mayneedtousemorecellssinceyouindicatedthesignalintheundilutedsampleisverylow.]
-washwith5mlCOLDPBS;resUSPendinice-cold5%MPA;[Fine.5%MPAcouldbeaddedtothesupernatantfordeproteination.]
-mixthoroughly,homogenizebysonication(2x5cycles),thencentrifugeat12.00rpmfor5minat4°C;[Fine]
-collectsupernatantforGSHassay;storeoniceORat-80°C.[NeedtoneutralizetheMPAtopH4~6,thenanalyzetheundilutedsamplewiththekit]
