The2-ABlabelingkitcontainstwosetsofthefollowingreagents(15samplesperkit)suppliedinglassampoulessealedunderpurenitrogen:  2-Aminobenzamide(2-ABdye)  Dimethylsulfoxide(DMSO)  Aceticacid  Sodiumcyanoborohydrideor2-picolineborane

TrADItional2-AAand2-ABlabellingkitsusesodiumcyanoborohydrideasareducingagentduringglycanlabeling.Thisreagentistoxicsoafumecupboardshouldbeusedduringhandling.ToconformwithemerginghealthandsafetyregulationswearenowreplacingthesewithournewVPglycankitsthatusepicolineboranewhichisasignificantlysaferreductant.

NumberofSamplesOne2-ABlabelingkitcontainsreagentstolabelupto30separateanalyticalsamplesperkit

Dyepurity>99%byHPLC

Molecularweight137

LamBDa-ex320nm

Lambda-em420nm

AmountofSampleFrom25pmolupto25nmolglycanspersample./p>

SuitableSamplesAnypurifiedglycanswithfreereducingterminicanbelabeled.

StructuralIntegrityNodetectable(<2molepercent)lossofsialicacid,fucose,sulfate,orphosphate.

LabelingEfficiencyTypically>85%(dependentonsample).

LabelingSelectivityEssentiallystoichiometriclabeling.

Protocol1. PurifytheglycansLudgerCleanEB10cartidges(LC-EB10-A6)havebeendesignedforpurificationofglycansfromproteins,salts,anddetergents.

2.TransfersampletoreactionvialTheamountofsampleshouldbeintherange100picomoles–50nanomolesforaglycanpoolobtainedfromatypicalglycoprotein.Withasinglepureglycanaslittleas5picomolescanbelabeledanddetectedinsubsequentHPLCanalysis.SuitablereactionvialsincludesmallpolypropylenemicrocentrifugetubesandtubesforPCRwork.

3. DrythesamplesIdeally,samplesshouldbedriedusingacentrifugalevaporator.IfthisisnotpossIBLethenfreezedrying(lyophilization)canbeusedwithcaution(inparticular,ensurethatthesampledriestoasmall,compactmassattheverybottomofthevial).Donotsubjectsamplestohightemperatures(>28°C)orextremesofpHastheseconditionswillresultinacidcatalysedlossofsialicacids(hightemperatures,lowpH)orepimerizationoftheglycanreducingterminus(athighpH).

4.PrepareaDMSO-aceticacidmixtureAdd150μLglacialaceticacidtothevialofDMSOandmixbyPipetteaction.

5.AddthedyeAdd100μLoftheDMSO-aceticacidmixturetoavialofthe2-AB(2-AminobenzamideAcid)dyeandmixuntilthedyeisdissolved.

6.AddthereductantAddthedissolveddyetoavialofsodiumcyanoborohydrideorpicolineboraneandmixbypipetteactionuntilthereductantiscompletelydissolvedtomakethefinallabelingreagent.

7.AddlabelingreagenttosamplesAdd5μLoflabelingreagenttoeachdriedglycansample,capthemicrotube,mixthoroughly.

8.IncubatePlacethereactionvialsinaheatingblock,sandtray,ordryovensetat65°Candincubatefor3hours.Inmostcases,theincubationtimecanbeshortenedto2hoursorextendedupto4hourswithoutsignificantlychangingtheoutcomeofthelabelingreaction.

9.CentrifugeandcoolAftertheincubationperiodremovethesamples,centrifugethemicrotubesbriefly,thenallowthemtocoolcompletelytoroomtemperature.

10.SampleCleanupPost-labelingsamplecleanupisrecommendedtoremoveexcessdyeandotherlabelingreagents.CleanupcanbeachievedusingLudgerCleanT1cartridges(LC-T1-A6)orScartridges(LC-S-A6)

2-ABLabelingReferences

AnumulaKR,DhumeST.Highresolutionandhighsensitivitymethodsforoligosaccharidemappingandcharacterizationbynormalphasehighperformanceliquidchromatographyfollowingderivatizationwithhighlyfluorescentanthranilicacid.GlycoBIOLOGy8685-694(1998)

BiggeJC,PatelTP,BruceJA,GouldingPN,CharlesSM,ParekhRB.Nonselectiveandefficientfluorescentlabelingofglycansusing2-aminobenzamideandanthranilicacid.AnalBiochem230:229-238(1995)

Guile,G.R.;Rudd,P.M.;Wing,D.R.;Prime,S.B.;Dwek,R.A.Arapidandhigh-resolutionhigh-performanceliquidchromatographicmethodforseparatingglycanmixturesandanalyzingoligosaccharideprofilesAnalyticalBiochemistry240:210-226(1996)

Hardy,M.R.‘Glycanlabelingwiththefluorophores2-aminobenzamideandanthranilicacid’in‘TechniquesinGlycobiology’,editedbyTownsend,R.RandHotchkiss,A.T..MarcelDekkerInc,NewYork(1997)

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