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    當前位置 ： NEB >>> NEB/Protein Deglycosylation Mix II/P6044S/20 reactions
    NEB/Protein Deglycosylation Mix II/P6044S/20 reactions
    • NEB/Protein Deglycosylation Mix II/P6044S/20 reactions

    NEB/Protein Deglycosylation Mix II/P6044S/20 reactions

    價格: ￥5880.00 市場價: 9800.00
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    貨號: P6044S-20reactions
    品牌: NEB
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      • Description:

        Glycosylationisoneofthemostcommonpost-translationalmodificationsofproteins,asshowninFigure1.N-linkedglycosylationoccurswhenglycansareattachedtoasparagineresiduesonthecoreprotein.O-linkedglycosylationoccurswhenglycansareattachedtoserineorthreonineresidues.Bothchemicalandenzymaticmethodsexistforremovingoligosaccharidesfromglycoproteins.However,chemicalmethodssuchasβ-eliminationwithmildalkaliormildhydrazinolysiscanbeharshandmayresultinincompletesugarremovalanddegradationoftheprotein;whereas,enzymaticmethodsaremuchgentlerandcanprovidecompletesugarremovalwithnoproteindegradation.

        PNGaseFisthemosteffectiveenzymaticmethodforremovingalmostallN-linkedoligosaccharidesfromglycoproteins.PNGaseFdigestiondeaminatestheaspargineresiduetoasparticacid,andleavestheoligosaccharideintact,keepingitsuitableforfurtheranalysis.Oligosaccharidescontainingafucoseα(1-3)-linkedtotheglycancoreare,however,resistanttoPNGaseFwhichcanoccuronsomeplantandinsectglycoproteins.

        ToremoveO-linkedglycans,monosaccharidesmustberemovedbyaseriesofexoglycosidasesuntilonlytheGalβ1-3GalNAc(core1)and/ortheGlcNAcβ1-3GalNAc(core3)coresremainattachedtotheserineorthreonine.NEB’sO-Glycosidase,clonedfromEnterococcusfaecalis,canthenremovethesecorestructureswithnomodificationoftheserineorthreonineresidues.Anymodificationofthecorestructures,includingsialyation,willblocktheactionoftheO-Glycosidase.Sialicacidresiduesareeasilyremovedbyageneralα2-3,6,8,9NeuraminidaseA.Inaddition,exoglycosidasessuchasβ(1-4)GalactosidaseSandβ-N-Acetylhexosaminidasefcanbeincludedindeglycosylationreactionstoremoveothercomplexmodificationsoftenknowntobepresentonthecorestructures.ThiscombinationofenzymesmaynotremoveallO-linkedoligosaccharidesbutshouldremovemanycommonoligosaccharidestructures.

        TheProteinDeglycosylationMixIIcontainsalloftheenzymes,reagents,andcontrolsneededtoremoveallN-linkedandsimpleO-linkedglycansaswellassomecomplexO-linkedglycans.Thismixcontainsenzymesufficientfor20reactionsorthecleavageofasmuchas2mgofglycoprotein.AlloftheenzymesandreagentsincludedintheProteinDeglycosyationMixIIareMassSpectrometrycompatIBLe.Followingthedeglycosylationreaction,samplesarereadytobepreparedformassspectrometryanalysis.

        Figure1:AGlycoproteinmodifiedwithO-linkedandN-linkedglycosylation.

        P6044_figure_1
        Figure2


        EnzymaticDeglycosylationofBovineFetuinunderbothnative(10XDeglycosylationMixBuffer1)andreducing(10XDeglycosylationMixBuffer2)conditions.20μgreactionswereloadedontoa10-20%Tris-glycineSDS-PAGEgel.
        Lane1:???ColorPrestainedProteinStandard,BroadRange(11-245?kDa)(NEB#P7712)
        Lane2:???20?μguntreatedFetuincontrol
        Lane3:???20μgFetuindeglycosylatedundernativeconditionswithDeglycosylationMixBuffer1
        Lane4:???20μgFetuindeglycosylatedunderreducingconditionswithDeglycosylationMixBuffer2
        Lane5:???5μlProteinDeglycosylationMixII.

        ProteinDeglycosylationMixII:
        PNGaseF(Glycerol-free),Recombinant:
        10,000units/vial

        O-Glycosidase:
        80,000units/vial

        α2-3,6,8,9NeuraminidaseA:
        400units/vial

        β1-4GalactosidaseS:
        960units/vial

        β-N-acetylhexosaminidasef:
        300units/vial

        SubstrateControl:
        Fetuin,0.5mg(FetuincontainssialylatedN-linkedandO-linkedglycans)

        ofEnzymesIncludedintheProteinDeglycosylationMixII
        O-Glycosidase(NEB#P0733),alsoknownasEndo-α-N-Acetylgalactosaminidase,isarecombinantenzymeclonedfromEnterococcusfaecalis(1).Itcatalyzestheremovalofcore1andcore3O-linkeddisaccharidesfromglycoproteins.Themolecularweightisapproximately147kDa.

        PNGaseF(Glycerol-free),Recombinant(NEB#P0709),alsoknownasPeptide:N-glycosidaseF,isclonedfromElizabethkingiamiricola(formerlyFlavobacteriummeningosepticum)andexpressedinE.coli(2).PNGaseF(Glycerol-free),RecombinantisanamidasewhichcleavesbetweentheinnermostGlcNAcandasparagineresiduesofhighmannose,hybrid,andcomplexoligosaccharidesfromN-linkedglycoproteinsunlessα(1-3)corefucosylated.Themolecularweightisapproximately36kDa.

        α2-3,6,8,9NeuraminidaseA(NEB#P0722),alsoknownasSialidaseA,isarecombinantenzymeclonedfromArthrobacterureafaciensandexpressedinE.coli(3).Itcatalyzesthehydrolysisofα2,3,α2,6,α2,8andα2,9linkedN-acetylneuraminicacidresiduesfromglycoproteinsandoligosaccharides.Themolecularweightisapproximately100kDa.

        β1-4GalactosidaseS(NEB#P0745),isarecombinantenzymeclonedfromStreptococcuspneumoniaeandexpressedinE.coli(4).Itisahighlyspecificexoglycosidasethatcatalyzesthehydrolysisofβ1-4linkedgalactoseresiduesfromoligosaccharides.Themolecularweightisapproximately231kDa.

        β-N-Acetylhexosaminidasef(NEB#P0721),isarecombinantenzymeclonedfromStreptomycesplicatus(5)andoverexpressedinE.coli(6).Itcatalyzesthehydrolysisofterminalβ-N-acetylgalactosamineandglucosamineresiduesfromoligosaccharides.Themolecularweightisapproximately100kDa.

        ReagentsSupplied

        Thefollowingreagentsaresuppliedwiththisproduct:

        Storeat(°C)Concentration
        DeglycosylationMixBuffer1-2010X
        DeglycosylationMixBuffer2-2010X

        Notes:

        TheProteinDeglycosylationMixIIissuppliedwithtwodifferentreactionbuffers.Pleasereferencethe“ProtocolsandManuals”tabforthespecificprotocolassociatedwitheachbuffersystem.The10XDeglycosylationMixBuffer1(NEB#B6044)shouldbeusedwhennative(non-denaturing)conditionsarenecessary.The10XDeglycosylationMixBuffer2(NEB#B6045)willreducetheglycoprotein,butwillalsoprovidethemostefficientandcompletelevelofdeglycosylation.Ifnon-denaturingconditionsarenotrequired,werecommendusing10XDeglycosylationMixBuffer2.Storage:Itisrecommendedtostorethiskitat4°C.Allcomponentsofthekitwillbestableforatleastoneyearifstoredcorrectly.TheProteinDeglycosylationMixIIisnotrecommendedforuseonMucin-likesubstrates.
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