CA-G055 | |
Name | EliteTM Glutathione(GSH)AssayKit(Fluorescence) |
Description | TheEliteTMGlutathioneAssayKitprovidesanultrasensitivefluorimetricassaytoquantifyGSHinsample.Theproprietarynon-fluorescentglutathionesensorusedinthekitbecomesstronglygreenfluorescentuponreactingwithaGSHcompound,whichhasthespectralpropertiesalmostidenticaltothoseoffluoresceinandcanbeeasilyreadbyafluorescencemicroplatereaderatEx/Em=490/520nm.Thekitcandetectaslittleas1picomoleofGSHina100μLassayvolume(10nM).Inaddition,bothabsorptionandemissionspectraoftheglutathioneadductarepH-independent,makingthisassaykithighlyrobust. |
Application | Theassaycanbeperformedinaconvenient96-wellor384-wellmicrotiter-plateformatandeasilyadaptedtoautomationwithoutaseparationstep. |
Size | 200assays |
Detection | Fluorescencemicroplatereader |
Clickherefor DATASHEET | Clickherefor MSDS |
FrequentlyAskedQuestions:
1.Q:Whatwouldyousuggesttopreparethecellsamplesforthisassay?
Answer:Allthedetergentsinvariouslysisbufferswillcausebackgroundnoisefortheassay.ThetrADItionalwaytodoitisthe"freezeandthaw"method(i.e.freezefor20-30minutesat-80Cthenthawatroomtemperature,2-3times),butitistootedious.Wefoundthat0.2%SDSgivesthebestresultsamongallthedetergentstested.
2.Q:DoesthisKitworkforplantsamples?Couldyourecommendaspecialprotocoltomeasuregluthationeinplants?Answer:Aslongasthesamplchop2gofplantparts(leaf,root,grain,etc.)andgrounditinahomogenizerfor2minwith5-10mlof0.1NHCl;centrifugetopbtaintheGSHcontainingsupernatant,thenadjustpHto6.5fortheGSHassay.
3.Q:Mysampleisfrozenbraintissue,howshouldIprepareitforthesaasy?
Answer:ItisbetterusePBS,andsimplyhomogenizeit,thenusethesupernatant.
4.Q:Iworkwithhumanprimarycells.HowmanycellsshouldIuseintheassay?
Answer:Suggesttostartwith10,000cells/wellfirst.
5.Q:Forthefluorescentdetection,anemissionfilterwith520nmisrecommended.Butwehaveonly535nmfilter.Woulditworkforthismeasurement?
Answer:Yes.
6.Q:Wedon"thavethefiltersfor490/520(nm).Whatwehavearefor395/535(nm).Woulditwork?
Answer:No.theemissionisOK,buttheexcitationisnot.Thelowestexcitationwavelengthisat460nm.
7.Q: Couldyoutakealookatmyprotocolforpreparingthecellculturelysatefortheassayandgiveyoursuggestion?
Answer:
-removesupernatant,thenwashwithHEPES-BSS;[Fine]
-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]
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