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主营:研究并生产荧光和发光探针,信号转导研究的试剂
℡ 4000-520-616
℡ 4000-520-616
AAT Bioquest/TF4-VAD-FMK/13471/25 Tests
产品编号:13471
市  场 价:¥27560.00
场      地:美国(厂家直采)
联系QQ:1570468124
电话号码:4000-520-616
邮      箱: info@ebiomall.com
美  元  价:$1378.00
品      牌: AAT Bioquest
公      司:AAT Bioquest
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AAT Bioquest/TF4-VAD-FMK/13471/25 Tests
商品介绍
Overview
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Ex/Em(nm)588/610
MW849.94
CAS#N/A
SolventDMSO
StorageF/D/L
CategoryEnzymeDetection
PeptidasesandProteases
RelatedCellApoptosis
ApoptosisandCytotoxicity
BiochemicalAssays
TF4-VADisaredfluorescentcell-permeablepolycaspaseinhibitortotargetcaspases1,2,3,6,8,9,or10.Onceinsidethecell,theinhibitorbindscovalentlytotheactivecaspase,whichproducesagreenfluorescence.Whenaddedtoapopulationofcells,theTF4-VAD-FMKprobeenterseachcellandcovalentlybindstoareactivecysteineresiduethatresidesonthelargesubunitoftheactivecaspaseheterodimer,therebyinhibitingfurtherenzymaticactivity.Theboundlabeledreagentisretainedwithinthecell,whileanyunboundreagentwilldiffuseoutofthecellandiswashedaway.Thegreenfluorescentsignalisadirectmeasureoftheamountofactivecaspasepresentinthecellatthetimethereagentwasadded.Cellsthatcontaintheboundlabeledreagentcanbeanalyzedby96-wellplate-basedfluorometry,fluorescencemicroscopy,orflowcytometry.TheprobehasthespectralpropertiessimilartoTexasRed®,thusthefiltersetofTexasRed®canbeconvenientlyusedwithaflowcytometerormicroscope.
SpectrumAdvancedSpectrumViewer

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Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

1.      GeneralSolutionCaspaseAssaysUsingAMC,AFC,pNA,R110andProRedSubstrates

1.1.    Preparea10mMstocksolutioninDMSO.

1.2.    Preparea2Xcaspase substrate(50µM)assaysolutionasthefollowing:

50µLsubstratestocksolution

100 µLDTT(1M)

400 µLEDTA(100mM)

10mLTrisBuffer(20mM),pH=7.4

1.3.    Mixequalvolumeofthecaspasestandardsorsampleswith2Xcaspasesubstrateassaysolution(fromStep1.2),andincubatethesolutionsatroomtemperatureforatleast1hour.

1.4.    Monitorthefluorescenceusingafluorescencemicroplatereader,orabsorbanceusinganabsorbancemicroplatereader.

2.      CellCaspaseAssaysUsingCell-PermeableFMKCaspaseProbes

2.1.    Preparea2-5mMstocksolutioninDMSO.

2.2.    Treatcellsasdesired.

2.3.    Preparea2Xpermeablecaspasesubstrate(20µM)assaysolutionbydilutingtheDMSOstocksolution(fromStep2.1)inHankswith20mMHepesbuffer(HHBS).

2.4.    Mixequalvolumeofthetreatedcellswith2Xcaspasesubstrateassaysolution(fromStep2.3),andincubatethecellsina37°C,5%CO2incubatorforatleast1hour.

2.5.    WashthecellswithHHBSforatleastonce.

2.6.    Monitorthefluorescenceintensitybyaflowcytometer,afluorescencemicroscopeorafluorescencemicroplatereader.

3.      CellCaspaseAssaysUsingCell-PermeableFMKCaspaseProbes(For#13470-13476only)

3.1.    Preparea250Xstocksolutionbyadding50µLDMSOintothevial.

3.2.    Treatcellsasdesired.

3.3.    Add250XDMSOstocksolution(fromStep3.1)intothecellsolutionata1:250ratio(suchas2uLto500uLcells),andincubatethecellsina37°C,5%CO2incubatorfor1hour.

3.4.    WashthecellswithHHBSforatleastonce.

3.5.    Monitorthefluorescenceintensitybyflowcytometer,fluorescencemicroscopyorfluorescentmicroplatereader.

References&Citations
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1.   LawrenceCP,ChowSC.(2012)SuppressionofhumanTcellproliferationbythecaspaseinhibitors,z-VAD-FMKandz-IETD-FMKisindependentoftheircaspaseinhibitionproperties.ToxicolApplPharmacol.

2.   LiYY,YanCL.(2012)[InhibitionofelicitationofallergiccontactdermatitisbytopicaluseofZ-VAD-FMK,abroadcaspaseinhibitor:experimentinmice].ZhonghuaYiXueZaZhi,92,1992.

3.   MeslinB,BeavoguiAH,FaselN,PicotS.(2011)PlasmodiumfalciparummetacaspasePfMCA-1triggersaz-VAD-fmkinhibitableproteasetopromotecelldeath.PLoSOne,6,e23867.

4.   MullerI,LamersMB,RitchieAJ,DominguezC,Munoz-SanjuanI,KiselyovA.(2011)Structureofhumancaspase-6incomplexwithZ-VAD-FMK:Newpeptidebindingmodeobservedforthenon-canonicalcaspaseconformation.BioorgMedChemLett,21,5244.

5.   AbaamraneL,RaffinF,SchmerberS,SendowskiI.(2011)Intracochlearperfusionofleupeptinandz-VAD-FMK:influenceofantiapoptoticagentsongunshot-inducedhearingloss.EurArchOtorhinolaryngol,268,987.

6.   EquilsO,Moffatt-BlueC,IshikawaTO,SimmonsCF,IlievskiV,HirschE.(2009)Pretreatmentwithpancaspaseinhibitor(Z-VAD-FMK)delaysbutdoesnotpreventintraperitonealheat-killedgroupBStreptococcus-inducedpretermdeliveryinapregnantmousemodel.InfectDisObstetGynecol,2009,749432.

7.   LiYY,YanCL,XuW.(2008)[AttenuationofallergiccontactdermatitisbyZ-VAD-FMK,abroadcaspaseinhibitor:experimentwithmice].ZhonghuaYiXueZaZhi,88,3153.

8.   WitteMD,DescalsCV,deLavoirSV,FloreaBI,vanderMarelGA,OverkleeftHS.(2007)Bodipy-VAD-Fmk,ausefultooltostudyyeastpeptideN-glycanaseactivity.OrgBiomolChem,5,3690.

9.   HengBC,ClementMV,CaoT.(2007)CaspaseinhibitorZ-VAD-FMKenhancesthefreeze-thawsurvivalrateofhumanembryonicstemcells.BiosciRep,27,257.

10.   VejuxA,LizardG,TourneurY,RiedingerJM,FrouinF,KahnE.(2007)Effectsofcaspaseinhibitors(z-VAD-fmk,z-VDVAD-fmk)onNileRedfluorescencepatternin7-ketocholesterol-treatedcells:investigationbyflowcytometryandspectralimagingmicroscopy.CytometryA,71,550.


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