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AAT Bioquest/SMCC Plus™ *Enhanced water solubility, crosslinking efficiency and stability*/4503/5 mg
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | None/None |
| MW | 465.37 |
| CAS# | N/A |
| Solvent | DMSO |
| Storage | F/D |
| Category | ProteinBiochemistry Generalproteins |
| Related | AminetoThiol Thiol-ReactiveProbes BiochemicalAssays |
Generally,a10-to50-foldmolarexcessofSMCCPlus™overtheamountofamine-containingproteinresultsinsufficientmaleimideactivationtoenableseveralsulfhydryl-containingproteinstobeconjugatedtoeachamine-containingprotein.Morediluteproteinsolutionsrequiregreaterfoldmolarexcessofreagenttoachievethesameactivationlevel.Empiricaltestingisnecessarytodetermineoptimalactivationlevelsandfinalconjugationratiosfortheintendedapplication.
1.Prepareconjugationbufferforamine-containingprotein,Protein-NH2(BufferA):Phosphatebufferedsaline(PBS,pH7.2)orotheramine-freebufferswithpH7.2to8.0areOKtouseforthispurpose.
Note:Avoidbufferscontainingprimaryamines(e.g.Trisorglycine)andsulfhydrylsduringconjugationbecausetheywillcompetewiththeintendedreaction.Ifnecessary,dialyzeordesaltsamplesintoanappropriatebuffersuchasPBS.
2.Prepareconjugationbufferforsulfhydryl-containingprotein,Protein-SH(BufferB):Sulfhydryl-freebuffersatpH6.5-7.5areOKtouseforthispurpose.
Note1:Avoidbufferscontainingsulfhydrylsduringconjugationbecausetheywillcompetewiththeintendedreaction.Ifnecessary,dialyzeordesaltsamplesintoanappropriatebuffersuchasPBS.
Note2:Theadditionof1-5mMEDTAhelpstochelatedivalentmetals,therebyreducingdisulfideformationinthesulfhydryl-containingprotein.
3.Preparedesaltingcolumntoseparatemodifiedproteinfromexcesscrosslinkerandreactionby-products.
4.Prepareamine-containing(Protein-NH2)andsulfhydryl-containingprotein(Protein-SH)solutionsintheirConjugationBuffersAandB.
5.PrepareProtein#1-SMCCPlus™conjugate:AddtheappropriateamountofSMCCPlus™toProtein#1solutioninbufferA.TheconcentrationoftheProtein-NH2determinesthemolarratioofSMCCPlus™required.TherequiredamountofSMCCPlus™issuggestedasfollows,butthebestratiohastobedeterminedexperimentallybyusingthetargetproteins:
§ Proteinsamples<1mg/mLuse40-80-foldmolarexcess.
§ Proteinsamplesof1-4mg/mLuse20-foldmolarexcess.
§ Proteinsamplesof5-10mg/mLuse5-to10-foldmolarexcess.
6.Incubatetheabovereactionmixtureatroomtemperaturefor30-60minutesorat4°Cfor2-4hours.
Note1:Tostoptheconjugationreactionbeforecompletion,addbuffercontainingreducedcysteineataconcentrationseveraltimesgreaterthanthesulfhydrylsofProtein-SH.
Note2:Conjugationefficiencycanbeestimatedbyelectrophoresisseparationandsubsequentproteinstaining.
7.RemoveexcessSMCCPlus™usingadesaltingcolumnequilibratedwithConjugationBufferA.
8.Combineandmixsulfhydryl-containing(Protein-SH)anddesaltedamine-containingprotein(Protein-NH2)-SMCCPlus™conjugateinamolarratiocorrespondingtothatdesiredforthefinalconjugateandconsistentwiththerelativenumberofsulfhydrylandactivatedaminesthatexistonthetwoproteins.
Note:Moleculestobereactedwiththemaleimidemoietymusthavefree(reduced)sulfhydryls.Forproteins,reducedisulfidebondsusing5mMTCEPatroomtemperaturefor30minutes,followedbytwopassesthroughasuitabledesaltingcolumn.Beawarethatproteins(e.g.,antibodies)maybeinactivatedbycompletereductionoftheirdisulfidebonds.Selectivereductionofhinge-regiondisulfidebondsinIgGcanbeaccomplishedwith2-mercaptoethylamine•HCl(2-MEA).SulfhydrylsmaybeaddedtomoleculesusingN-succinimidylS-acetylthioacetate(SATA)or2-iminothiolane•HCl(Traut’sReagent),whichmodifyprimaryamines.
| References&Citations |  CitationExplorer |
N-glycantargetedgenedeliverytothedendriticcellSIGNreceptor
Authors:KevinAnderson,ChristianFernandez,KevinGRice
Journal:Bioconjugatechemistry(2010):1479--1485
