Appropriateuseofindividualglycosidasesallowthedeterminationofsialyation(useofSialidaseonly)andthespecificpresenceofN-linkedglycans(usingPNGaseFonly)andO-linkedglycans(usingSialidase,Beta-Galactosidase,O-Glycosidase,andGlucosaminidase).Alternatively,QA-Bioproducesapremixedsolutionoftheenzymesinone20microlitrevialinourDeGlycoMxKit(KE-DGMX). |
Includes20microlitreseachofthefollowingenzymes:
- PNGaseF(Chryseobacteriummeningosepticum)
- O-Glycosidase(Streptococcuspneumoniae)
- Sialidase(Arthrobacterureafaciens)
- Beta-Galactosidase(Streptococcuspneumoniae)
- Glucosaminidase(Streptococcuspneumonia)
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OtherSuppliedReagents:- Reactionbuffer
- DenaturationSolution
- TritonX
- BovineFetuin(control)
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 |
Specificity
TheEnzymaticCarboReleaseKitwilldeglycosylateglycoproteins,removingallN-linkedoligosaccharidesandmanyO-linkedoligosaccharidesfromglycoproteins.N-links(Asparganine-linked)areremovedusingtheenzymePNGaseF.Inaddition,allSerineorThreoninelinked(O-linked)Gal-(b1-3)-GalNAc-(a1)andallsialicacidsubstitutedGal-(b1-3)-GalNAc-(a1)willberemovedusingthecombinationofSialidaseandO-Glycosidase.TheadditionofBeta-GalactosidaseandGlucosaminidasewillassistinthedeglycosylationoflargerO-linkstructures. |
KitCapacity
Thequantityofenzymesrecommendedintheprotocolsissufficienttodeglycosylateapproximately100μgofanaverageglycoproteininthetimegiven.PNGaseFcleavageisgenerallytheratelimitingreactionduetotheslowremovalofsomestericallyhinderedN-linkedresidues,evenwhentheglycoproteinisdenatured.Sincealloftheenzymesretainactivityunderreactionconditionsforseveraldays,amuchlargerquantityofglycoproteinmaybedeglycosylatedifincubationisextended.Conversely,thereisnoneedtousetherecommendedamountsofenzymesifquantitiesmuchlessthan100μgofglycoproteinarebeingcleaved.Theenzymescanbedilutedinto1XReactionBuffer7.Theywillremainstableindilutedformat4°C.
BovineFetuinControlProtein
BovineFetuincontainssialylatedN-andO-linkedoligosaccharides13.
NOTE:CommercialpreparationsofFetuincontainproteaseswhichwilleventuallydegradetheprotein.TheFetuinControlhasbeenheattreatedat90°Cfor10minutestoinactivatetheproteases.TheFetuinsolutioncanbestoredat4°C.
DenaturingProtocol
1.Dissolve100μgorlessofaglycoproteinin30μLdeionizedwaterinanEppendorftube.
2.Add10μL5XReactionBuffer7and2.5μLDenaturationSolution.Mixgently.
3.Heatat100°Cfor5minutes.
NOTE:SomeproteinsmayprecipitatewhenheatedwithSDS.Inthisevent,omittheheattreatmentandincreasetheincubationtimeto24hoursafteraddingenzymes.
4.Cooltoroomtemperature.Add2.5μlTritonX-100solution.Mixgently.
NOTE:FailuretoaddTritonX-100mayresultinthereductionofactivityofsomeenzymes.
5.Add1μLeachofeachenzyme.
6.Incubatefor3hoursat37°C.
7.Analyzebymethodofchoice.
Alternatively,theenzymesmaybeaddedindividuallyorsequentiallyinordertodeterminewhattypesofoligosaccharidesarepresentontheglycoprotein.
Non-denaturingProtocol
1.Dissolve100μgorlessofaglycoproteinin35μLdeionizedwaterinanEppendorftube.
2.Add10μL5XReactionBuffer7.
3.Add1μLofeachenzyme.
4.Incubatefor1-5daysat37°C.
Analiquotshouldbedeglycosylatedusingthedenaturingprotocoltoprovideagelstandardforthefullydeglycosylatedprotein.Thepositionofthenativeproteincanthenbecomparedwiththisstandardtojudgetheextentofdeglycosylation.
ReferencestoDeglycosylateGlycoproteins
Kobata,A.Useofendo-andexoglycosidasesforstructuralstudiesofglycoconjugates.AnalBiochem100:1-14(1979).
KimMS,LeahyD.Enzymaticdeglycosylationofglycoproteins.MethodsEnzymol.533:259-63(2013).
MagnelliPE,BielikAM,GuthrieEP.Identificationandcharacterizationofproteinglycosylationusingspecificendo-andexoglycosidases.JVisExp.Dec26;(58):e3749(2011).
SeguZM,HusseinA,NovotnyMV,MechrefY.AssigningN-glycosylationsitesofglycoproteinsusingLC/MSMSinconjunctionwithendo-M/exoglycosidasemixture.JProteomeRes.Jul2;9(7):3598-607(2010).
Sojar,H.T.andO.P.Bahl.Achemicalmethodforthedeglycosylationofproteins.ArchBiochemBiophys259:52-57(1987).
TarentinoA.L.andT.H.Plummer.Enzymaticdeglycosylationofasparagine-linkedglycans:purification,properties,andspecificitlyofoligosaccharide-cleavingenzymesfromFlavobacteriummeningosepticum.MethodsinEnzymol230:44-57(1994).
Uchida,Y.,Y.TsukadaandT.Sugimori.EnzymaticpropertiesofneuraminidasesfromArthrobacterureafaciens.JBiochem(Tokyo)86:573-585(1979).
ZhangW,WangH,ZhangL,YaoJ,YangP.Large-scaleassignmentofN-glycosylationsitesusingcomplementaryenzymaticdeglycosylation.Talanta.Jul15;85(1):499-505(2011).
品牌介绍
QA-bio公司是一家致力于提供糖基化研究工具的专业公司，所提供的糖基化研究产品涵盖糖苷内/外切酶，去糖基化酶，唾液酸产品，多糖产品纯化试剂盒，多糖标记试剂盒，HPLC柱及其缓冲液，同时公司还提供多糖分析服务。此外，公司还提供部分分子生物学产品。
