The2-AAlabelingkitcontainstwosetsofthefollowingreagents(15samplesperkit)suppliedinglassampoulessealedunderpurenitrogen:
2-aminobenzoicacid(2-AAdye)
Dimethylsulfoxide(DMSO)
Aceticacid
Sodiumcyanoborohydrideor2-picolineborane

TrADItional2-ABand2-AAlabellingkitsusesodiumcyanoborohydrideasareducingagentduringglycanlabeling.Thisreagentistoxicsoafumecupboardshouldbeusedduringhandling.ToconformwithemerginghealthandsafetyregulationswearenowreplacingthesewithournewVPglycankitsthatusepicolineboranewhichisasignificantlysaferreductant.

NumberofSamplesOne2-AAlabelingkitcontainsreagentstolabelupto30separateanalyticalsamplesperkit.

Dyepurity>99%byHPLC

Molecularweight137

LamBDa-ex320nm

Lambda-em420nm

AmountofSampleFrom25pmolupto25nmolglycanspersample.

SuitableSamplesAnypurifiedglycanswithfreereducingterminicanbelabeled.

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

LabelingEfficiencyTypically>85%(dependentonsample).

LabelingSelectivityEssentiallystoichiometriclabeling.

Protocol
1. Purifytheglycans
LudgerCleanEB10cartidges(LC-EB10-A6)havebeendesignedforpurificationofglycansfromproteins,salts,anddetergents.

2.Transfersampletoreactionvial
Theamountofsampleshouldbeintherange100picomoles–50nanomolesforaglycanpoolobtainedfromatypicalglycoprotein.Withasinglepureglycanaslittleas5picomolescanbelabeledanddetectedinsubsequentHPLCanalysis.SuitablereactionvialsincludesmallpolypropylenemicrocentrifugetubesandtubesforPCRwork.

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

4.PrepareaDMSO-aceticacidmixture
Add150μLglacialaceticacidtothevialofDMSOandmixbyPipetteaction.

5.Addthedye
Add100μLoftheDMSO-aceticacidmixturetoavialofthe2-AA(2-aminobenzoicacid)dyeandmixuntilthedyeisdissolved.

6.Addthereductant
Addthedissolveddyetoavialofsodiumcyanoborohydrideorpicolineboraneandmixbypipetteactionuntilthereductantiscompletelydissolvedtomakethefinallabelingreagent.

7.Add2-AAlabelingreagenttosamples
Add5μLoflabelingreagenttoeachdriedglycansample,capthemicrotube,mixthoroughly.

8.Incubate
Placethereactionvialsinaheatingblock,sandtray,ordryovensetat65°Candincubatefor3hours.Inmostcases,theincubationtimecanbeshortenedto2hoursorextendedupto4hourswithoutsignificantlychangingtheoutcomeofthelabelingreaction.

9.Centrifugeandcool
Aftertheincubationperiodremovethesamples,centrifugethemicrotubesbriefly,thenallowthemtocoolcompletelytoroomtemperature.

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

2-AALabelingReferences

Anumula,K.R.Quantitativedeterminationofmonosaccharidesinglycoproteinsbyhigh-performanceliquidchromatographywithhighlysensitivefluorescencedetection.AnalyticalBiochemistry220:275-283(1994)

Anumula,KR;Dhume,STHighresolutionandhighsensitivitymethodsforoligosaccharidemappingandcharacterizationbynormalphasehighperformanceliquidchromatographyfollowingderivatizationwithhighlyfluorescentanthranilicacid.GlycoBIOLOGy8:685-694(1998)

Anumula,KR;Du,PCharacterizationofcarbohydratesusinghighlyfluorescent2-aminobenzoicacidtagfollowinggelelectrophoresisofglycoproteins.AnalyticalBiochemistry275:236-242(1999)

Bigge,JC;Patel,TP;Bruce,JA;Goulding,PN;Charles,SM;Parekh,RBNonselectiveandefficientfluorescentlabelingofglycansusing2-aminobenzamideandanthranilicacid.AnalyticalBiochemistry230:229-238(1995)

Frears,ER;Merry,AH;Axford,JSScreeningneutralandacidicIgGN-glycansbyhighdensityelectrophoresis.GlycoconjugateJournal16:283-290(1999)

Huang,Z;Prickett,T;Potts,M;Helm,RFTheuseofthe2-aminobenzoicacidtagforoligosaccharidegelelectrophoresis.CarbohydrateResearch328:77-83(2000)

Sato,K;Sato,K;Okubo,A;Yamazaki,SDeterminationofmonosaccharidesderivatizedwith2-aminobenzoicacidbycapillaryelectrophoresis.strong>AnalyticalBiochemistry251:119-121(1997)