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Ex/Em(nm)	None/None
MW	266.21
CAS#	55750-62-4
Solvent	DMSO
Storage	F/D
Category	ProteinBiochemistry Generalproteins
Related	AminetoThiol Thiol-ReactiveProbes BiochemicalAssays

3-MaleimidopropionicacidN-hydroxysuccinimideesterisanon-cleavableandmembranepermeablecrosslinker.Itcontainsanamine-reactivesuccinimidylester(SE)andasulfhydryl-reactivemaleimidegroup.NHSestersreactwithprimaryaminesatpH7-9toformstableamidebonds.MaleimidesreactwithsulfhydrylgroupsatpH6.5-7.5toformstablethioetherbonds.TheSEgroupof3-maleimidopropionicacidN-hydroxysuccinimideesterisreactedwithlysineresiduesonthecarrierprotein,convertingthemtoreactivemaleimides.3-MaleimidopropionicacidN-hydroxysuccinimideesterisareagentthatiswidelyusedforgeneratingstablemaleimide-activatedcarrierproteinsthatcanspontaneouslyreactwithsulfhydryls.Alternativelytheserelativelystablemaleimide-activatedintermediatesmaybelyophilizedandstoredforlaterconjugationtoahapten.

Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

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.

References&Citations

CitationExplorer

N-glycantargetedgenedeliverytothedendriticcellSIGNreceptor
Authors:KevinAnderson,ChristianFernandez,KevinGRice
Journal:Bioconjugatechemistry(2010):1479--1485

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