Description:
The M30Apoptosense®CK18Kit (ELISA)isauniquetoolforreliableandreproducIBLequantitativedetectionofapoptosisinepithelialkeratin18(K18) (cytokeratin18[CK18])positivecells.ThespecificityisreliablebecausetheM30Apoptosense®ELISAsolelyquantifiestheaccumulationofsolublecaspase-cleavedkeratin18,aproductofapoptosis.Theassayisintendedforsubjectserumorplasma. Anotherusefulapplicationisthedetectionoftumorapoptosisinmousexenograftmodelsusingplasmasamples.
M30Apoptosense®isanELISAassaybasedontheM30monoclonalantibody(Leersetal.,1999).Thisantibodyspecificallybindscaspase-cleavedkeratin18(ccK18,caspase-celavedcytokeratin18,ccCK18orK18-Asp396)butdoesnotbindnative,uncleaved,K18.TheM30Apoptosense®assaythereforerepresentsaspecificquantitativetestforapoptosisinepithelialcells(Häggetal.2002).
Allreagentsareprovidedinaconvenientready-for-useformat.Theseparablestripsprovidetheflexibilityforyourexperimentaldesign.
Becauseofthecriticalimportanceofapoptosis,numerousapplicationfieldsexists,butparticularly:
- Hepatology:Researchshowsthatapoptosisisincreasedinliverdisease.CK18ishighlyabundantandcriticalfornormallivers.Apoptosiscanbemeasuredinliverdiseasesubjects.
- Oncology:Measureschemotherapyinducedapoptosisinepithelialcarcinomainresearch
- Toxicology:Suitableforstudieswhenevaluatingdifferentpharmaceuticalagents’impactonlivercelldeath
Note:M30Apoptosense®ELISAwaspreviouslysoldundercatalog#P10010.
CK18KitAdvantages:
- Onlymethodavailabletospecificallymeasureepithelialapoptosisintheblood
- SpecificquantificationmeasurementtoolforapoptosisinCK18positivecells
- SandwichELISAina96wellplateinaconvenientready-to-useformat
- Easytoperform,onlyaminimumofpipettingstepsrequired
- Itcanbesplitupforuseatseveraloccasions
- CK18ispresentinsimple epithelialcellsonly,thusincreasingspecificityofmeasurementinepithelial-basedmodels
- RemarkablestABIlityoftheCK18proteincomplexesinthecirculation,thusprovidingstablestorageofserum/plasmasamplesandallowingformultiplefreezethawcycles.
- Minimalday-to-dayfluctuationsinhealthysubjects
- SuitabletousetogetherwithM65®ELISAforquantificationofapoptosis,necrosisandtotalcelldeath
CK18KitComposition:
- M5CoatedMicrostrips:Onemicroplate,12stripswith8wellseach,96drywells intotal.ThewellsarecoatedwithmousemonoclonalK18antibodyM5.The microplateissealedinanaluminiumbag,whichcontainsadesiccatingdevice.If notallthestripsareused,resealthebagandkeepthedesiccatingdeviceinside. Readyforuse!
- M30Conjugate:Concentrate(24×conc).Onevialcontaining0.4mLofmouse monoclonalM30antibody(anti-K18Asp396neo-epitope)conjugatedwith horserADIshperoxidase(HRP)inphosphatebufferwithproteinstabilizers. Preservativeadded.ShouldbedilutedwithM30ConjugateDilutionBuffer. Note:Donotexposetolight.
- M30ConjugateDilutionBuffer:Onevialcontaining11mLofphosphatebuffer withproteinstabilizersfordilutionoftheM30Conjugate.Preservativeadded. Bluecolored.
- M30StandardA–G:StandardAcontaining2mLofphosphatebufferwithfetal calfserum(FCS).StandardB–G,0.5mLeach,containingstandardmaterialin phosphatebufferwithFCS.ThevaluesofStandardA–Gare0,75,150,250,500, 750and1000U/L,respectively.Preservativeadded.Yellowcolored.Readyfor use!StandardAcanbeusedfordilutionsofsamples>1000U/L.
- M30ControlLow&High:Twovialscontaining0.5mLofreactivecomponents inphosphatebufferwithFCS.ThevaluesofM30ControlLowandM30Control Higharestatedontherespectivevials.Preservativeadded.Yellowcolored. Readyforuse!
- WashTablet:Onetabletfor500mLofpreparedwashsolution.Dissolvethe WashTabletin500mLoffreshdeionizedwater.
- TMBSubstrate:Onebottlecontaining22mLofTMB(3,3’,5,5’-Tetramethylbenzidine) Solution.Note!Donotexposetolight!Readyforuse!
- StopSolution:Onevialcontaining7mLof1.0Msulphuricacid.Readyforuse!
- SealingTape:One(1)sheet.
MeasurementPrinciple:
TheM30ApoptosenseELISAisasolid-phasesandwichenzymeimmunoassay. Standards,controlsandsamplesreactwithasolidphasecaptureantibodyM5 directedagainstCK18andtheHRP-(horseradishperoxidase)conjugatedM30 antibodydirectedagainsttheCK18Asp396neo-epitope.Unboundconjugateis removedbyawashingstep.TMBSubstrateisadded.Thecolordevelopment isstoppedandtheabsorbanceisread.Theresultingcolorisdirectly proportionaltotheconcentrationoftheanalyte. Byplottingastandardcurvefromknownconcentrationsversusmeasured absorbance,theamountofantigeninthesamplecanbecalculated.The concentrationoftheantigenisexpressedasunitsperliter(U/L).
Thekitcontainscalibratorsandcontrols,andcanbeusedinanylaboratory.Calibratorsandcontrolscontainrecombinantpeptides,whichincludeM5andM30epitopes.Thetestcanprovidearesultwithin5 hours.StandardELISAequipmentisusedandthustheM30kitoffersandeasytouseformatthatshouldallowwideaccessibilityfortesting.
InXenograftModels
TheM30 Apoptosense®ELISA detectshuman,butnotmouse/rat,CK18.ThedetectionofCK18inthebloodofamousecarryingahumantumorxenograftisthereforeduetoapoptosisofthehumantumorcells. Olofssonet.al(CancerBioMarkers,2009)usedaslightlymodifiedprotocolallowingforlessvolume(12.5µlsample)andusedanadditionalblockingagentaddedtotheconjugate(ie,HRPPlus).
Background:
DevelopmentoftheM30antibodyintoanELISArepresentedthefirstresearchtooltomeasureaspecificapoptosisproductintheblood.TheM30Apoptosense®ELISAmeasuresthelevelsofsolubleccK18fragmentscontainingtheK18Asp396neo-epitope.Afterinductionofapoptosisinepithelialtissues,ccK18increasesareobservedintheblood.Caspase-cleavedK18fragmentsareremarkablystableinhumanserum,furtheraddingtoconvenienceandtheeasytousemethodofthisELISA.
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谢谢了
要原因
请问有什么方法检测细胞内活性氧更好，各位有什么经验，及相关试剂盒推荐一下。
请问下有无同学需要H37RA的？我是做EAE模型的，上个月购买了BDDifco公司的H37RA（货号），因为购买的时候只能整盒6支购买，但我们用不了那么多，所以想问问有无同学需要的，100mg/支，800元/支或用等价试剂交换。地址广州。有需要的请私信，谢谢！
（1）优级纯试剂 亦称保证试剂，为一级品，纯度高，杂质极少，主要用于精密分析和科学研究，常以GR表示。
（2）分析纯试剂 亦称分析试剂，为二级品，纯度略低于优级纯，杂质含量略高于优级纯，适用于重要分析和一般性研究工作，常以AR表示。
（3）化学纯试剂 为三级品，纯度较分析纯差，但高于实验试剂，适用于工厂、学校一般性的分析工作，常以CP表示。
（4）实验试剂 为四级品，纯度比化学纯差，但比工业品纯度高，主要用于一般化学实验，不能用于分析工作，常以 LR表示。
以上按试剂纯度的分类法已在我国通用。根据化学工业部颁布的“化学试剂包装及标志”的规定，化学试剂的不同等级分别用各种不同的颜色来标志，见表1。
表1 我国化学试剂的等级及标志
