AAT Bioquest/Sulfo-SMCC [4-(N-Maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester, sodium salt]

Overview

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Generally,a10-to50-foldmolarexcessofSMCCorSMCCPlus™overtheamountofamine-containingproteinresultsinsufficientmaleimideactivationtoenableseveralsulfhydryl-containingproteinstobeconjugatedtoeachamine-containingprotein.Morediluteproteinsolutionsrequiregreaterfoldmolarexcessofreagenttoachievethesameactivationlevel.Empiricaltestingisnecessarytodetermineoptimalactivationlevelsandfinalconjugationratiosfortheintendedapplication.

1.Prepareconjugationbufferforamine-containingprotein,Protein-NH₂(BufferA):Phosphatebufferedsaline(PBS,pH7.2)orotheramine-freebufferswithpHfrom7.2to8.0areOKtouseforthispurpose.

Note:Avoidbufferscontainingprimaryamines(e.g.,Trisorglycine)andsulfhydrylsduringconjugation,becausetheywillcompetewiththeintendedreaction.Ifnecessary,dialyzeordesaltsamplesintoanappropriatebuffersuchasPBS.

2.Prepareconjugationbufferforsulfhydryl-containingprotein,Protein-SH(BufferB):Sulfhydryl-freebuffersatpHfrom6.5to7.5areOKtouseforthispurpose.

Note1:Avoidbufferscontainingsulfhydrylsduringconjugation,becausetheywillcompetewiththeintendedreaction.Ifnecessary,dialyzeordesaltsamplesintoanappropriatebuffersuchasPBS.

Note2:Theadditionof1-5mMEDTAhelpstochelatedivalentmetals,therebyreducingdisulfideformationinthesulfhydryl-containingprotein.

3.Preparedesaltingcolumntoseparatemodifiedproteinfromexcesscrosslinkerandreactionby-products.

4.Prepareamine-containingprotein(Protein-NH₂)andsulfhydryl-containingprotein(Protein-SH)solutionsintheirConjugationBufferAandBufferB.

5.PrepareProtein1-NH₂-SMCC(orSMCCPlus™)conjugate:AddtheappropriateamountofSMCC(orSMCCPlus™)totheamine-containingproteinsolutioninBufferA.TheconcentrationoftheProtein-NH₂determinesSMCC(orSMCCPlus™)molarexcesstouse.ThesuggestedvolumeofSMCC(orSMCCPlus™)molarexcessesareasfollows,butthebestratiohastobedeterminedexperimentallyusingthetargetproteins:

•Proteinsamples<1mg/mLuse40-80-foldmolarexcess.

•Proteinsamplesof1-4mg/mLuse20-foldmolarexcess.

•Proteinsamplesof5-10mg/mLuse5-to10-foldmolarexcess.

6.Incubatetheabovereactionmixtureatroomtemperaturefor30-60minutesorat4°Cfor2-4hours.

Note1:Tostoptheconjugationreactionbeforecompletion,addbuffercontainingreducedcysteineataconcentrationseveraltimesgreaterthanthatofthesulfhydrylsofProtein-SH.

Note2:Conjugationefficiencycanbeestimatedbyelectrophoresisseparationandsubsequentproteinstaining.

7.RemoveexcessSMCC(orSMCCPlus™)byusingadesaltingcolumnequilibratedwithConjugationBufferA.

8.Combineandmixthiol-containing(Protein-SH)anddesaltedamine-containingprotein(Protein-NH₂)SMCC(orSMCCPlus™)conjugateinamolarratiocorrespondingtothatdesiredforthefinalconjugateandconsistentwiththerelativenumberofsulfhydrylandactivatedaminesthatexistonthetwoproteins.

Note:Moleculestobereactedwiththemaleimidemoietymusthavefree(reduced)sulfhydryls.Forproteins,reducedisulfidebondsusing5mMTCEPatroomtemperaturefor30minutes,followedbytwopassesthroughasuitabledesaltingcolumn.Beawarethatproteins(e.g.,antibodies)maybeinactivatedbycompletereductionoftheirdisulfidebonds.Selectivereductionofhinge-regiondisulfidebondsinIgGcanbeaccomplishedwith2-mercaptoethylamine•HCl(2-MEA).SulfhydrylscanbeaddedtomoleculesusingN-succinimidylS-acetylthioacetate(SATA)or2-iminothiolane•HCl(Traut’sReagent),whichmodifyprimaryamines.

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