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)