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    當前位置 : NEB >>> NEB/SNAP-Cell? Starter Kit/E9100S/1 set
    NEB/SNAP-Cell? Starter Kit/E9100S/1 set
    • NEB/SNAP-Cell? Starter Kit/E9100S/1 set

    NEB/SNAP-Cell? Starter Kit/E9100S/1 set

    價格: ¥3156.00 市場價: 5260.00

    貨號: E9100S-1set
    品牌: NEB
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      • Description:

        TheSNAP-tag?isanoveltoolforthespecific,covalentattachmentofvirtuallyanymoleculetoaproteinofinterest,providingsimplicityandextraordinaryversatilitytotheimagingofproteinsinliveandfixedcells,andtothestudyofproteinsinvitro.Thecreationofasinglegeneconstructyieldsataggedfusionproteincapableofformingacovalentlinkagetoavarietyoffunctionalgroups,includingfluorophores,biotin,orbeads.Thissystemprovidesapowerfulanduniquetooltostudytheroleofproteinsinavarietyofhighlydynamicprocesses,includingproteintrafficking,turnover,andcomplexformation.?

        TheSNAP-tagisa20kDamutantofthehumanDNArepairproteinO6-alkylguanine-DNAalkyltransferase(hAGT)thatreactsspecificallyandrapidlywithbenzylguanine(BG)andbenzylchloropyrimidine(CP)derivatives,leADIngtocovalentlabelingoftheSNAP-tagwithasyntheticprobe(Figure1).TheSNAP-taghasanumberoffeaturesthatmakeitidealforavarietyofproteinlabelingapplications.TherateofthereactionoftheSNAP-tagwiththesederivativesislargelyindependentofthenatureofthesyntheticprobeattachedtoBG,permittingthelabelingofSNAPfusionproteinswithawidevarietyoffunctionalgroups.ManyoftheseSNAP-tagsubstratesarecell-permeable,allowinglive-cellimagingofproteinexpressionandlocalization(Figure2).TheABIlitytoturnonthesignalatwill,togetherwiththeavailabilityofacell-permeablenonfluorescentblockingagent(SNAP-Cell?Block),allowstime-resolvedpulse-chaseanalysisofproteintrafficking.Finally,theavailabilityoforthogonalproteinlabelingsystemsfromNEBpermitssimultaneouslabelingofmultipleproteinsinasinglecell(CLIP-tag?,aSNAP-tagvariantthatreactsexclusivelywithO2-benzylcytosinesubstrates,andtheACP/MCPtags,smallproteintagswhichcanbeenzymaticallylabeledonthecellsurfacewithCoenzymeAderivatives).

        TheSNAP-CellStarterKitcontainsamammalianexpressionplasmid(pSNAPf)encodingtheSNAP-tagflankedbyrestrictionsitesforcloningageneofinterest,andtwocell-permeablefluorescentSNAP-tagsubstrates.Apositivecontrolplasmid(pSNAPf-Cox8A),encodingaSNAP-taggedprotein(cytochromecoxidase)withawell-characterizedmitochondriallocalization,isalsoincluded.Lastly,anegativecontrol“blockingagent”(SNAP-CellBlock)isincludedthatinteractswiththeSNAP-tag,butisnotfluorescent.Therearetwostepstousingthissystem:subcloningandexpressionoftheproteinofinterestasaSNAPffusion,andlabelingofthefusionwiththeSNAP-tagsubstrateofchoice.

        Figure1:SNAP-tagReactionFigure 1: SNAP-tag Reaction

        Figure2:?LivecellimagingofSNAPfFusionProteins.Figure 2. Live cell imaging of SNAPf Fusion proteins

        LiveCHO-K1cellstransientlytransfectedwithpSNAPf-H2B.CellswerelabeledwithSNAP-Cell505-star(green)for15minutesat37°C,5%CO2.
        Figure3:?LivecellimagingofSNAPfFusionProteins.
        Figure 3: Live cell imaging of SNAPf Fusion Proteins
        LiveCOS-7cellsexpressingmitochondrialcytochromeoxidase8A-SNAPf(SNAPf-Cox8A)werelabeledwithSNAP-CellTMR-Star(red).NucleiwerecounterstainedwithHoechst33342(blue).

        KitComponents

        Thefollowingreagentsaresuppliedwiththisproduct:

        Storeat(°C)Concentration
        pSNAPfVector-200.5mg/ml
        pSNAPf-Cox8AControlPlasmid-200.5mg/ml
        SNAP-Cell®505-Star10nmol
        SNAP-Cell®TMR-Star-206nmol
        SNAP-Cell®Block-2020nmol

        Notes:

        Forlong-termstorage,allkitcomponentsshouldbestoredat-20?C.Plasmidsolutionscanbestoredat4?Cforuptooneweek.Undissolveddyeandblockingsubstratescanbestoredat4?Cforupto4weeksprotectedfromlightandmoisture.Withproperstorageat-20°Cthesubstratesshouldbestableforatleastthreeyearsdryor3monthsdissolvedinDMSO.NEB10-betaCompetentE.coli(HighEfficiency)(NEB#C3019)isrecommendedforpropagatingandsubcloningofthevectorandcontrolplasmid.
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