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AAT Bioquest/Sulfo-SMCC [4-(N-Maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester, sodium salt]
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| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | None/None |
| MW | 436.37 |
| CAS# | 92921-24-9 |
| Solvent | DMSO |
| Storage | F/D |
| Category | ProteinBiochemistry Generalproteins |
| Related | AminetoThiol Thiol-ReactiveProbes BiochemicalAssays |
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
