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).

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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)	OtherSuppliedReagents: Reactionbuffer DenaturationSolution TritonX BovineFetuin(control)
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.