SMCC-结构反应解释

8.3ASMCC是一类含有N-羟基琥珀酰亚胺(NHS)活性酯和马来酰亚胺的双功能偶联剂•可以将分别含有巯基和氨基的化合物键接在一起。NHS活性酯与伯胺在PH7-9的环境形成酰胺键。马来酰胺与巯基在的环境下形成稳定的硫醚键。在水溶液中，NHS活性酯的水解是(与氨基的反应)个竞争反应。马来酰胺比NHS稳定，但是在PH大于时，马来酰胺会慢慢水解，失去与巯基反应的特异性。因而,在使用SMCC时通常是在的环境下进行，并且先让NHS发生反应。SMCC结构里的环己烷环可以降低马来酰胺的水解速率。这使得蛋白质在用SMCC修饰之后可以冻干存放一段时间。很多蛋白质都选用该试剂来进行马来酰亚胺修饰。用SMCC来制备抗体-酶或者半抗原作载体的蛋白质，经常采用两步合成法。首先，含有氨基的蛋白质与几倍的偶联剂反应，反应结束后通过脱盐柱或者透析的方法除掉没有反应玩的SMCC。然后，再与含有巯基的蛋白质反应。在实际操作中要注意的是，SMCC怕潮湿，存放时要和干燥剂一起存放。并且使用中从冰箱拿出来时要先在室外放置一段时间平衡温度，以免立刻开启，空气中水分遇冷凝结，破坏SMCC结构。INSTRUCTIONS

SMCC(succinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate),50mgMolecularWeight:SpacerArm:ANetMassAdded:Storage:Uponreceiptstoredesiccatedat4°C.Productisshippedatambienttemperature.Sulfo-SMCC(sulfosuccinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate),1gSulfo-SMCC,50mgSulfo-SMCC,No-WeighFormat,8X2mgmicrotubesMolecularWeight:SpacerArm:ANetMassAdded:CAS#:92921-24-9Storage:Uponreceiptstoredesiccatedat-20°C.ProducSulfo-SMCC(sulfosuccinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate),1gSulfo-SMCC,50mgSulfo-SMCC,No-WeighFormat,8X2mgmicrotubesMolecularWeight:SpacerArm:ANetMassAdded:CAS#:92921-24-9Storage:Uponreceiptstoredesiccatedat-20°C.Productisshippedatambienttemperature.IntroductionSMCCanditswater-solubleanalogSulfo-SMCCareheterobifunctionalcrosslinkersthatcontainN-hydroxysuccinimide(NHS)ticdegradationisacompetingreactionwhoserateincreaseswithpH.ThemaleimidegroupismorestablethantheNHS-estergroupbutwillslowlyhydrolyzeandlosesitsreactionspecificityforsulfhydrylsatpHvalues>.Forthesereasons,conjugationswiththesecrosslinkersareusuallyperforesterandmaleimidegroupsthatamine-andsulfhydryl-containingwithprimaryaminesatpH7-9maleimidesreactwithsulfhydrylablethioetherbonds.Inaqueousmolecules.NHSestersreactesterandmaleimidegroupsthatamine-andsulfhydryl-containingwithprimaryaminesatpH7-9maleimidesreactwithsulfhydrylablethioetherbonds.Inaqueousmolecules.NHSestersreacttoformamidebonds,whilegroupsatpHtoformstsolutions,NHSesterhydrolyallowcovalentconjugationofmedatpHwiththeNHS-ester(amine-targeted)reactedbeforeorsimultaneouswiththemaleimide(sulfhydryl-targeted)reaeorsimultaneouswiththemaleimide(sulfhydryl-targeted)reaction.ThecyclohexaneringinthespacerarmofthesereagentsdecreasestherateofhydrolysisofthemaleimidegroupcomparedtosimilarreagentsthatdonotcontainthisThisfeatureenablesproteinsthathavebeenmaleimide-activatedwithSMCCorSulfo-SMCCtobelyophilizedandstoredforlaterconjugationtoasulfhydryl-containingmolecule.Manymaleimide-activatedproteinproductsareproducedinthismanner(seeRelatedProducts).ThecyclohexaneringinthespacerarmofthesereagentsdecreasestherateofhydrolysisofthemaleimidegroupcomparedtosimilarreagentsthatdonotcontainthisThisfeatureenablesproteinsthathavebeenmaleimide-activatedwithSMCCorSulfo-SMCCtobelyophilizedandstoredforlaterconjugationtoasulfhydryl-containingmolecule.Manymaleimide-activatedproteinproductsareproducedinthismanner(seeRelatedProducts).SMCCandSulfo-SMCCareoftenusedtoprepareantibody-enSMCCandSulfo-SMCCareoftenusedtoprepareantibody-enzymeandhapten-carrierproteinconjugatesinatwo-stepreactionscheme.First,theamine-containingproteinisreactedwzymeandhapten-carrierproteinconjugatesinatwo-stepreactionscheme.First,theamine-containingproteinisreactedwithbyremovalofexcess(nonreacted)reagentbydesaltingordialysis;finally,thesulfhydryl-containingmoleculeisaddedtoreactwiththemaleimidegroupsalreadyattachedtothefirstprotein.Sulfo-SMCCissolubleinwaterandmanyotheraqueousbufferstoapproximately10mM,althoughsolubilitydecreaseswithincreasingsaltconcentration.SMCCisnotdirectlywater-solubleandmustbedissolvedinanorganicsolventsuchasdimethylsulfoxide(DMSO)ordimethylformamide(DMF);subsequentdilutionintoaqueousreactionbufferisgenerallypossible,andmostproteinreactantswillremainsolubleifithbyremovalofexcess(nonreacted)reagentbydesaltingordialysis;finally,thesulfhydryl-containingmoleculeisaddedtoreactwiththemaleimidegroupsalreadyattachedtothefirstprotein.Sulfo-SMCCissolubleinwaterandmanyotheraqueousbufferstoapproximately10mM,althoughsolubilitydecreaseswithincreasingsaltconcentration.SMCCisnotdirectlywater-solubleandmustbedissolvedinanorganicsolventsuchasdimethylsulfoxide(DMSO)ordimethylformamide(DMF);subsequentdilutionintoaqueousreactionbufferisgenerallypossible,andmostproteinreactantswillremainsolubleifthefinalconcentrationoforganicsolventislessthan10%.aseveral-foldmolarexcessofthecrosslinker,followedSMCCandSulfo-SMCCImportantProductInformationSMCCandSulfo-SMCCaremoisture-sensitive.Storereagentvialindesiccant.EquilibratevialtoroomtemperaturebeforeopeningtoavoidmoisturecondensationinsidethecontainerDissolveneededamountofreagentanduseitimmediatelybeforehydrolysisoccurs.Discardanyunusedreconstitutedreasolution.gent.Donotstorereagentinaltsamplesintoanappropriatebuffersuchasphosphate-bufferedsaline(PBS).solution.No-WeighMicrotubeHandling:Immediatelybeforeuse,puncturethemicrotubefoilwithapipettetip,add200lof50mMsodiumphosphatebuffer(pHorultrapurewaterandpipetteupanddowntomix.Afteruse,cuttheusedmicrotubefromthemicrotubestripanddiscard.StoretheunusedmicrotubesinthefoilNo-WeighMicrotubeHandling:Immediatelybeforeuse,puncturethemicrotubefoilwithapipettetip,add200lof50mMsodiumphosphatebuffer(pHorultrapurewaterandpipetteupanddowntomix.Afteruse,cuttheusedmicrotubefromthemicrotubestripanddiscard.Storetheunusedmicrotubesinthefoilpouchprovided.Note:Donotusephosphate-bufferedsaline(PBS)forinitNote:Donotusephosphate-bufferedsaline(PBS)forinitialdissolutionofSulfo-SMCC;thereagentdoesnotdissolveialdissolutionofSulfo-SMCC;thereagentdoesnotdissolvewellinbuffersexceeding50wellinbuffersexceeding50mMtotalsalts.However,oncedissolved,thesolutioncanbefurtherdilutedinPBSordissolved,thesolutioncanbefurtherdilutedinPBSorothernon-aminebuffers.AvoidbufferscontainingprimaryaminesTrisorglycine)andsulfhydrylsduringconjugation,becausetheywillcompothernon-aminebuffers.AvoidbufferscontainingprimaryaminesTrisorglycine)andsulfhydrylsduringconjugation,becausetheywillcompetewiththeintendedreaction.Ifnecessary,dialyzeordesetewiththeintendedreaction.Ifnecessary,dialyzeordesMoleculestobereactedwiththemaleimidemoietymusthavefree(reduced)sulfhydryls.ReducepeptidedisulfidebondswithImmobilizedTCEPDisulfideReducingGel(ProductNo.77712).Forproteins,reducedisulfidebondsusing5mMTCEP(1:100dilutionofBond-BreakerTCEPSolution,ProductNo.77720)for30minutesatroomtemperature,followedbytwopassesthroughasuitabledesaltingcolumn.,ZebaDesaltSpinColumns).Beawarethatproteins.,antibodies)maybeinactivatedbycompletereductionoftheirdisulfidebonds.Selectivereductionofhinge-regiondisulfidebondsinIgGcanbeaccomplishedwith2-Mercaptoethylamine・HCl(2-MEA,ProductNo.20408).SulfhydrylscanbeaddedtomoleculesusingN-succinimidylS-acetylthioacetate(SATA,ProductNo.26102)or2-iminothiolane・HCl仃raut'sReagent,ProductNo.26101),whichmodifyprimaryamines.ProcedureforTwo-stepProteinCrosslinkingGenerally,a10-to50-foldmolarexcessofcrosslinkerovertheamountofamine-containingproteinresultsinsufficientmaleimideactivationtoenableseveralsulfhydryl-containingproteinstobeconjugatedtoeachamine-containingprotein.Morediluteproteinsolutionsrequiregreaterfoldmolarexcessofreagenttoachievethesameactivationlevel.Empiricaltestingisnecessarytodetermineoptimalactivationlevelsandfinalconjugationratiosfortheintendedapplication.A.MaterialPreparation•ConjugationBuffer:phosphate-bufferedsaline(PBS=100mMsodiumphosphate,150mMsodiumchloride,pH;.,ProductNo.28372)orotheramine-andsulfhydryl-freebufferatpH(seeImportantProductInformation)-addingEDTAto15mMhelpstochelatedivalentmetals,therebyreducingdisulfideformationinthesulfhydryl-containingproteinDesaltingcolumntoseparatemodifiedproteinfromexcesscrosslinkerandreactionbyproducts.,ZebaDesaltSpinColumns)•Amine-containing(Protein-NH2)andsulfhydryl-containingproteins(Protein-SH)tobeconjugatedB.ProtocolNote:Forbestresults,ensurethatProtein-SHispreparedandreadytocombinewithProtein-NH2instep5.1.PrepareProtein-NH2inConjugationBuffer.Addtheappropriateamountofcrosslinkertotheproteinsolution.TheconcentrationoftheProtein-NH2determinesthecrosslinkermolarexcesstouse.Suggestedcrosslinkermolarexcessesareasfollows(alsoseeTable1):Proteinsamples<1mg/mluse40-80-foldmolarexcess.Proteinsamplesof1-4mg/mluse20-foldmolarexcessProteinsamplesof5-10mg/mluse5-to10-foldmolarexcess.

Table1.Crosslinkerpreparationandmolarexcesstousefor1mlofsample.Immediatelybeforeuse,dissolvecrosslinkerintheappropriatesolventattheconcentrationdenotedinparentheses;thenaddthelistedvolumetoa1mlproteinsample.Forexample,tousetheNo-WeighSulfo-SMCC,dissolvethe2mgcontentsofthemicrotubein200口lofbufferandthenaddtheprescribedvolumetoper1mlsample.Fortheotherproducts,theappropriateamountofdryrmgSulfo-SMCCforeagentmustbeweighedonabalancemgSulfo-SMCCfordissolutionin500口丨buffer).Protein-NH2Concentration(basedona50kDaprotein)10mg/ml1mg/mlmgProtein-NH2Concentration(basedona50kDaprotein)10mg/ml1mg/mlmg/ml(in50mMsodiumphosphateorwater)100卩lmg/ml*)40丨mg/ml*)50口丨mg/ml*)No-WeighSulfo-SMCC(in50mMsodiumphosphateorwater)50卩l(10mg/ml*)20口l(10mg/ml*)25|il(10mg/ml*)SMCC(inDMSOorDMF)100卩lmg/ml*)100卩lmg/ml*)100卩lmg/ml*)*ConcentrationofeachcrosslinkerbeforeaddingtoproteiExcess5X20XCrosslinkerMolar50XSulfo-SMCCnsample.Note:IftheSulfo-SMCCsolutiondoesnotcompletelydissolve,placethetubeunderhotrunningwaterorincubatefoolve,placethetubeunderhotrunningwaterorincubateforseveralminutesina50°Cwaterbath.Incubatereactionmixtureforrseveralminutesina50°Cwaterbath.Incubatereactionmixturefor30minutesatroomtemperatureor2eratureor2hoursat4°C.4.RemoveexcesscrosslinkerusingadesaltingcolumnequilibratedwithConjugationBuffer.5.CombineandmixProtein-SHanddesaltedProtein-NH2inamolarratiocorrespondingtothatdesiredforthefinalconjugateandconsistentwiththerelativenumberofsulfhydrylandactivatedaminesthatexistonthetwoproteins.6.Incubatethereactionmixtureatroomtemperaturefor3onminutesor2hoursatNote:Generally,theretoproceedforsevera4°C.isno4.RemoveexcesscrosslinkerusingadesaltingcolumnequilibratedwithConjugationBuffer.5.CombineandmixProtein-SHanddesaltedProtein-NH2inamolarratiocorrespondingtothatdesiredforthefinalconjugateandconsistentwiththerelativenumberofsulfhydrylandactivatedaminesthatexistonthetwoproteins.6.Incubatethereactionmixtureatroomtemperaturefor3onminutesor2hoursatNote:Generally,theretoproceedforsevera4°C.isnohoursharminallowingtheorovernight,althoughreactiusuallythereactionwillbecompleteinthespecifiedtime.Tolythereactionwillbecompleteinthespecifiedtime.Tostoptheconjugationreactionbeforecompletion,addbufferconcentrationseveraltimescontainingreducedcysteineataconcentrationseveraltimesgreaterthanthesulfhydrylsofProtein-SH.Note:ConjugatiogreaterthanthesulfhydrylsofProtein-SH.Note:Conjugationefficiencycanbeestimatedbyelectrophoresisseparationandsubsequentproteinstaining.nefficiencycanbeestimatedbyelectrophoresisseparationandsubsequentproteinstaining.AdditionalInformationAdditionalInformationA.PleasevisitthePiercewebsiteforadditionalinformationincludingthefollowingitem:A.PleasevisitthePiercewebsiteforadditionalinformationincludingthefollowingitem:TechTip:Attachanantibodyontoglass,silicaorquartTechTip:zsurfaceB.Two-stepreactionschemeMaleimide-activatedAntibodyAntibody-enzymeConjugateSulfo-SMCCAntibodyAntibodyAntibodyEnzymeEnzymeFigure1.Two-stepreactionschemeforconjugatingantibodyandenzymeproteinswithSulfo-SMCC.Inthisexample,thecrosslinkerisfirstreactedwiththeantibodytoproduceamaleimide-activatedprotein.Afterexcessnon-reactedcrosslinkerandby-productsareremoved,themaleimide-activatedantibodyisreactedwiththeappropriatemolarratioofenzymehavingsulfhydrylgroups.Usually,severalormultiplemaleimide-activationsoccurperantibodymolecule,enablingseveralenzymemoleculestobeconjugatedtoeachantibodymolecule.MBS/BDB/SMCC/sulfo-SMCC1、SMCC琥珀酰亚胺-4-（N-马来酰亚胺）环已烷-1-1羟酸酯分子一端的NHS酯基团与某一蛋白质分子的伯氨反应形成稳定的酰胺键，另一端（马来酰亚胺基团一