ProductName | GlutathioneDetectionKit |
Description | FluorescentdetectionoftotalGSHcontent |
SpeciesReactivity | SpeciesIndependent |
Platform | Microplate |
SampleTypes | Celllysates,EDTAPlasma,Erythrocytes,HeparinPlasma,Serum,Tissue,Urine,WholeBlood |
DetectionMethod | FluorometricAssay |
AssayType | DirectQuantitativeAssay |
Utility | FluorometricdetectionassaytomeasurethetotalGSHcontentinsamples. |
Sensitivity | 45nMintheFreeGSHand48nMintheTotalGSH |
AssayRange | 0.195-25uM |
Precision | IntraAssayPrecision:TwoeachofSSAtreatedhumanurineandwholebloodsampleswerefurtherdilutedin1%SSASampleDiluentandruninreplicatesof20inanassay.ThemeanandprecisionofthecalculatedGSHconcentrationswere:Sample1(Free)-1.27µM,4.0%CV,Sample1(Total)-2.30µM,4.7%CVSample2(Free)-2.00µM,3.1%CV,Sample2(Total)-3.80µM,4.7%CVSample3(Free)-8.33µM,4.6%CV,Sample3(Total)-9.77µM,2.7%CVSample4(Free)-3.89µM,3.0%CV,Sample4(Total)-4.45µM,2.3%CVInterAssayPrecision:TwoeachofSSAtreatedhumanurineandbloodsampleswerefurtherdilutedin1%SSASampleDiluentandruninduplicatesintwentyassaysrunovermultipledaysbytwooperators.ThemeanandprecisionofthecalculatedGSHconcentrationswere:Sample1(Free)-1.30µM,8.6%CV,Sample1(Total)-2.40µM,9.3%CVSample2(Free)-1.83µM,14.7%CV,Sample2(Total)-3.57µM,10.0%CVSample3(Free)-9.38µM,6.0%CV,Sample3(Total)-11.67µM,6.0%CVSample4(Free)-4.89µM,7.2%CV,Sample4(Total)-5.89µM,8.0%CV |
NumberofSamples | 39samplesinduplicate |
OtherResources | KitBooklet,MSDS |
StorageTemperature | 4ºC | |||||||||||||||||||||||||||
ShippingTemperature | BlueIce | |||||||||||||||||||||||||||
ProductType | DetectionKits | |||||||||||||||||||||||||||
AssayOverview | TheGlutathioneFluorescentDetectionkitisdesignedtoquantitativelymeasureglutathione(GSH),andoxidizedglutathione(GSSG)presentinavarietyofsamples.Thekitisuniqueinthatbothfreeandoxidizedglutathionearedetectedinthesamewellinthemicrotiterplate.Noseparationorwashingisrequired.TotalglutathioneisthesumofGSSGplusGSH.AGSHstandardisprovidedtogenerateastandardcurvefortheassayandallsamplesshouldbereadoffthestandardcurve.Thekitutilizesaproprietarynon-fluorescentmolecule,StressXpress®DetectionReagent,thatwillcovalentlybindtothefreethiolgrouponGSHtoyieldahighlyfluorescentproduct.AftermixingthesampleorstandardwiththeDetectionReagentandincubatingatroomtemperaturefor15minutes,thefluorescentproductisreadat510nminafluorescentplatereaderwithexcitationat390nm.TheconcentrationoftheGSHinthesampleiscalculated,aftermakingasuitablecorrectionforanydilutionofthesample,usingsoftwareavailablewithmostfluorescenceplatereaders.Freeglutathione,GSH,isreadfirstafter15minutes,followedbyadditionofareactionmixturethatconvertsalltheoxidizedglutathione,GSSG,intofreeGSH,whichthenreactswiththeexcessDetectionReagenttoyieldthesignalrelatedtoTotalGSHcontent.ThetotalconcentrationofGSHgeneratedinthesampleiscalculatedfromthegeneratedsignal.Wehaveprovideda96wellplateformeasurementbutthisassayisadaptableforhigherdensityplateformats.TheendusershouldensurethattheirHTSblackplateissuitableforusewiththesereagentspriortorunningsamples. | |||||||||||||||||||||||||||
KitOverview |
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CiteThisProduct | GlutathioneDetectionKit(StressMarqBiosciencesInc.,VictoriaBCCANADA,Catalog#SKT-202) |
AlternativeNames | γ-L-Glutamyl-L-cysteinylglycineDetectionKit(2S)-2-Amino-5-[[(2R)-1-(carboxymethylamino)-1-oxo-3-sulfanylpropan-2-yl]amino]-5-oxopentanoicacidDetectionKit |
ResearchAreas | Cancer,OxidativeStress |
ScientificBackground | Glutathione(L-γ-glutamyl-L-cysteinylglycine;GSH)isthehighestconcentrationnon-proteinthiolinmammaliancellsandispresentinconcentrationsof0.5-10mM(1).GSHplaysakeyroleinmanybiologicalprocesses,includingthesynthesisofproteinsandDNA,thetransportofaminoacids,andtheprotectionofcellsagainstoxidation.Harmfulhydrogenperoxidecellularlevelsareminimizedbytheenzymeglutathioneperoxidase(GP)usingGSHasareductant(2).TheoxidizedGSHdimer,GSSG,isformedfromGSHandperoxidebytheGPreaction(seebelow).AnimportantroleofGSSGintheNFγBactivatingsignalcascadeissuggestedbythefactsthatthepotentNFγBinducer,tetradecanoylphorbolacetate,increasesintracellularGSSGlevelsandGSSG/GSHratios(3).GlutathioneS-transferases(GST)areanimportantgroupofenzymesthatcatalyzethenucleophilicadditionofGSHtoelectrophiles.Theyareencodedby5genefamilies;4encodecytosolicGSTandoneencodesthemicrosomalformofGST.Theyhavebeenimplicatedinanumberofdiseases.InasthmaarachidonicacidisconvertedtounstableleukotrieneA4(LTA4).LTA4iseitherhydratedtoformLTB4oritisconjugatedtoGSHbyaGST,leukotrieneC4synthase,toformleukotrieneC4.LTC4anditsderivativeLTD4areimportantmoleculesinbronchialasthma.LeukotrieneC4synthaseisthereforeanimportanttherapeutictarget.IthasalsobeenshownthatincreasedexpressionofGSTscanleadtodrugresistance.Threeglutathioneadductsofthedrugmelphalan,usedtotreatovariancancerandmultiplemyeloma,havebeenisolatedfromreactionsinvolvinghumanmicrosomalGSTs. |
References | 1.MeisterA.(1988)TrendsBiochemSci.13(5):185-8. 2.MeisterA.(1994)JBiolChem.269(13):9397-400. 3.DrögeW.,etal.(1994)FASEBJ.8(14):1131-8. |
TypicalStandardCurveofGlutathioneDetectionKitStressXpress®–SKT-202.AssayType:Direct.DetectionMethod:FluorometricAssay.AssayRange:0.195–25uM
LinearitywasdeterminedbytakinghumanRBCsattwodifferentconcentrationsandmixedintheratiosgivenbelow.Themeasuredconcentrationswerecomparedtotheexpectedvaluesbasedontheratiosused.
Wepurchasedandcomparedapopularcolorimetrictotalglutathioneassaykit(kitT)thatusesEllmansreagenttodetectfreeglutathioneinthesample.Initialexperimentsusedrandomhumanurinesamplesthatwereprocessedasdescribedineachkitbooklet.WithkitT,thevaluesobtainedforurineaftertherecommendedtreatmentwith4volumesof5%metaphosphoricacidandsubsequent10folddilutionwithassaybufferputallthevalueswellbelowtheloweststandard.However,theurinesamplesrunintheStressXpress®kitgaveTotalGSHvaluesbetween0.63and4.04?M.
Currentlytherearenocitationsforthisproduct.
StressMarq Biosciences Inc.位于加拿大维多利亚,是一家生物科技公司,专门从事试剂与试剂盒研究。我们有强大的国际经销商网络,主要为私人和公众客户提供经我们多次试验成功的试剂,服务范围遍及全球40多个国家。 我们公司的核心技术领域为细胞应激与离子通道以及载体研究,同时在其他领域也取得了一定成就,包括翻译后修饰,提供甲基化与乙酰基化抗体。其中,细胞应激领域主要包括热休克蛋白(HSP)领域。我们公司不仅在热休克蛋白领域领先全球,而且在氧化应激领域也卓有成就。StressMarq的优势在于提供四种独立的产品系列,分别涉及抗体、蛋白、酶联免疫吸附试验(ELISA)试剂盒及小分子领域。 通过定位好以上四个领域,研究人员能够运用研究相关的整套试剂工具,将研究产品重点放在热休克蛋白70和90(HSP70和HSP90)两个核心领域。产品主要包括ELISA试剂盒、抑制剂、蛋白质(包括无内毒素制剂,而非低内毒素制剂)以及经过高级验证的抗体,产品在全球范围内均有销售。 StressMarq因其试剂验证质量过硬,跻身为高端公司。研究人员能放心使用抗体产品,因为本产品将具有免疫印迹(WB)、免疫沉淀、组织染色技术的功能,如免疫组织化学(IHC)和免疫细胞化学(ICC)。 本公司提供氧化应激产品,如8OHdG(DNA受损)ELISA试剂盒,同时还提供专门的抗体,适用于非定量技术,如免疫组化。在免疫组化中,组织的目标可视化更为重要。除了提供用于进一步研究的相关工具外,StressMarq还提供新的尖端研究工具,用于开发新的生物项目,包括全球独一无二的α B晶体蛋白ELISA试剂盒(现在是公认的乳腺癌标志物)以及热休克同源蛋白70(Hsc70)ELISA试剂盒。本公司已将专业技术扩展到抗体、小分子离子通道与载体等更新领域,前者包括当前热门的核心领域:Nav、Cav、TRP、Kv、KCNQ、HCN,后者包括当前热门的核心领域:ENaC、NCC和NKCC2以及主要水通道蛋白载体。