SMCC结构反应解释.docx

1、SMCC结构反应解释SMCC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PH6.5-7.5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7.5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PH7.2-7.5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

2、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。INSTRUCTIONSSMCC(succinimidyl4-N-maleimidomethylcyclohexane-1-carboxylate),50mgMolecularWe

3、ight:334.32SpacerArm:8.3NetMassAdded:219.09Storage:Uponreceiptstoredesiccatedat4C.Productisshippedatambienttemperature.Sulfo-SMCC(sulfosuccinimidyl4-N-maleimidomethylcyclohexane-1-carboxylate),1gSulfo-SMCC,50mgSulfo-SMCC,No-WeighFormat,82mgmicrotubesMolecularWeight:436.37SpacerArm:8.3NetMassAdded:21

4、9.09CAS#:92921-24-9Storage:Uponreceiptstoredesiccatedat-20C.Productisshippedatambienttemperature.IntroductionSMCCanditswater-solubleanalogSulfo-SMCCareheterobifunctionalcrosslinkersthatcontainN-hydroxysuccinimide(NHS)esterandmaleimidegroupsthatallowcovalentconjugationofamine-andsulfhydryl-containing

5、molecules.NHSestersreactwithprimaryaminesatpH7-9toformamidebonds,whilemaleimidesreactwithsulfhydrylgroupsatpH6.5-7.5toformstablethioetherbonds.Inaqueoussolutions,NHSesterhydrolyticdegradationisacompetingreactionwhoserateincreaseswithpH.ThemaleimidegroupismorestablethantheNHS-estergroupbutwillslowlyh

6、ydrolyzeandlosesitsreactionspecificityforsulfhydrylsatpHvalues7.5.Forthesereasons,conjugationswiththesecrosslinkersareusuallyperformedatpH7.2-7.5,withtheNHS-ester(amine-targeted)reactedbeforeorsimultaneouswiththemaleimide(sulfhydryl-targeted)reaction.Thecyclohexaneringinthespacerarmofthesereagentsde

7、creasestherateofhydrolysisofthemaleimidegroupcomparedtosimilarreagentsthatdonotcontainthisring.1Thisfeatureenablesproteinsthathavebeenmaleimide-activatedwithSMCCorSulfo-SMCCtobelyophilizedandstoredforlaterconjugationtoasulfhydryl-containingmolecule.Manymaleimide-activatedproteinproductsareproducedin

8、thismanner(seeRelatedProducts).SMCCandSulfo-SMCCareoftenusedtoprepareantibody-enzymeandhapten-carrierproteinconjugatesinatwo-stepreactionscheme.First,theamine-containingproteinisreactedwithaseveral-foldmolarexcessofthecrosslinker,followedbyremovalofexcess(nonreacted)reagentbydesaltingordialysis;fina

9、lly,thesulfhydryl-containingmoleculeisaddedtoreactwiththemaleimidegroupsalreadyattachedtothefirstprotein.Sulfo-SMCCissolubleinwaterandmanyotheraqueousbufferstoapproximately10mM,althoughsolubilitydecreaseswithincreasingsaltconcentration.SMCCisnotdirectlywater-solubleandmustbedissolvedinanorganicsolve

10、ntsuchasdimethylsulfoxide(DMSO)ordimethylformamide(DMF);subsequentdilutionintoaqueousreactionbufferisgenerallypossible,andmostproteinreactantswillremainsolubleifthefinalconcentrationoforganicsolventislessthan10%.SMCCandSulfo-SMCCImportantProductInformationSMCCandSulfo-SMCCaremoisture-sensitive.Store

11、reagentvialindesiccant.Equilibratevialtoroomtemperaturebeforeopeningtoavoidmoisturecondensationinsidethecontainer.Dissolveneededamountofreagentanduseitimmediatelybeforehydrolysisoccurs.Discardanyunusedreconstitutedreagent.Donotstorereagentinsolution.No-WeighMicrotubeHandling:Immediatelybeforeuse,pun

12、cturethemicrotubefoilwithapipettetip,add200lof50mMsodiumphosphatebuffer(pH7.0-7.5)orultrapurewaterandpipetteupanddowntomix.Afteruse,cuttheusedmicrotubefromthemicrotubestripanddiscard.Storetheunusedmicrotubesinthefoilpouchprovided.Note:Donotusephosphate-bufferedsaline(PBS)forinitialdissolutionofSulfo

13、-SMCC;thereagentdoesnotdissolvewellinbuffersexceeding50mMtotalsalts.However,oncedissolved,thesolutioncanbefurtherdilutedinPBSorothernon-aminebuffers.Avoidbufferscontainingprimaryamines(e.g.,Trisorglycine)andsulfhydrylsduringconjugation,becausetheywillcompetewiththeintendedreaction.Ifnecessary,dialyz

14、eordesaltsamplesintoanappropriatebuffersuchasphosphate-bufferedsaline(PBS).Moleculestobereactedwiththemaleimidemoietymusthavefree(reduced)sulfhydryls.ReducepeptidedisulfidebondswithImmobilizedTCEPDisulfideReducingGel(ProductNo.77712).Forproteins,reducedisulfidebondsusing5mMTCEP(1:100dilutionofBond-B

15、reakerTCEPSolution,ProductNo.77720)for30minutesatroomtemperature,followedbytwopassesthroughasuitabledesaltingcolumn(e.g.,ZebaDesaltSpinColumns).Beawarethatproteins(e.g.,antibodies)maybeinactivatedbycompletereductionoftheirdisulfidebonds.Selectivereductionofhinge-regiondisulfidebondsinIgGcanbeaccompl

16、ishedwith2-MercaptoethylamineHCl(2-MEA,ProductNo.20408).SulfhydrylscanbeaddedtomoleculesusingN-succinimidylS-acetylthioacetate(SATA,ProductNo.26102)or2-iminothiolaneHCl(TrautsReagent,ProductNo.26101),whichmodifyprimaryamines.ProcedureforTwo-stepProteinCrosslinkingGenerally,a10-to50-foldmolarexcessof

17、crosslinkerovertheamountofamine-containingproteinresultsinsufficientmaleimideactivationtoenableseveralsulfhydryl-containingproteinstobeconjugatedtoeachamine-containingprotein.Morediluteproteinsolutionsrequiregreaterfoldmolarexcessofreagenttoachievethesameactivationlevel.Empiricaltestingisnecessaryto

18、determineoptimalactivationlevelsandfinalconjugationratiosfortheintendedapplication.A.MaterialPreparationConjugationBuffer:phosphate-bufferedsaline(PBS=100mMsodiumphosphate,150mMsodiumchloride,pH7.2;e.g.,ProductNo.28372)orotheramine-andsulfhydryl-freebufferatpH6.5-7.5(seeImportantProductInformation)a

19、ddingEDTAto1-5mMhelpstochelatedivalentmetals,therebyreducingdisulfideformationinthesulfhydryl-containingproteinDesaltingcolumntoseparatemodifiedproteinfromexcesscrosslinkerandreactionbyproducts(e.g.,ZebaDesaltSpinColumns)Amine-containing(Protein-NH2)andsulfhydryl-containingproteins(Protein-SH)tobeco

20、njugatedB.ProtocolNote:Forbestresults,ensurethatProtein-SHispreparedandreadytocombinewithProtein-NH2instep5.1.PrepareProtein-NH2inConjugationBuffer.2.Addtheappropriateamountofcrosslinkertotheproteinsolution.TheconcentrationoftheProtein-NH2determinesthecrosslinkermolarexcesstouse.Suggestedcrosslinker

21、molarexcessesareasfollows(alsoseeTable1):Proteinsamples1mg/mluse40-80-foldmolarexcess.Proteinsamplesof1-4mg/mluse20-foldmolarexcess.Proteinsamplesof5-10mg/mluse5-to10-foldmolarexcess.Table1.Crosslinkerpreparationandmolarexcesstousefor1mlofsample.Immediatelybeforeuse,dissolvecrosslinkerintheappropria

22、tesolventattheconcentrationdenotedinparentheses;thenaddthelistedvolumetoa1mlproteinsample.Forexample,tousetheNo-WeighSulfo-SMCC,dissolvethe2mgcontentsofthemicrotubein200lofbufferandthenaddtheprescribedvolumetoper1mlsample.Fortheotherproducts,theappropriateamountofdryreagentmustbeweighedonabalance(e.

23、g.,2.4mgSulfo-SMCCfordissolutionin500lbuffer).Protein-NH2Concentration(basedona50kDaprotein)10mg/ml1mg/ml0.5mg/mlCrosslinkerMolarExcess5X20X50XSulfo-SMCC(in50mMsodiumphosphateorwater)100l(4.8mg/ml*)40l(4.8mg/ml*)50l(4.8mg/ml*)No-WeighSulfo-SMCC(in50mMsodiumphosphateorwater)50l(10mg/ml*)20l(10mg/ml*)

24、25l(10mg/ml*)SMCC(inDMSOorDMF)100l(3.7mg/ml*)100l(1.5mg/ml*)100l(1.8mg/ml*)*Concentrationofeachcrosslinkerbeforeaddingtoproteinsample.Note:IftheSulfo-SMCCsolutiondoesnotcompletelydissolve,placethetubeunderhotrunningwaterorincubateforseveralminutesina50Cwaterbath.3.Incubatereactionmixturefor30minutes

25、atroomtemperatureor2hoursat4C.4.RemoveexcesscrosslinkerusingadesaltingcolumnequilibratedwithConjugationBuffer.5.CombineandmixProtein-SHanddesaltedProtein-NH2inamolarratiocorrespondingtothatdesiredforthefinalconjugateandconsistentwiththerelativenumberofsulfhydrylandactivatedaminesthatexistonthetwopro

26、teins.6.Incubatethereactionmixtureatroomtemperaturefor30minutesor2hoursat4C.Note:Generally,thereisnoharminallowingthereactiontoproceedforseveralhoursorovernight,althoughusuallythereactionwillbecompleteinthespecifiedtime.Tostoptheconjugationreactionbeforecompletion,addbuffercontainingreducedcysteinea

27、taconcentrationseveraltimesgreaterthanthesulfhydrylsofProtein-SH.Note:Conjugationefficiencycanbeestimatedbyelectrophoresisseparationandsubsequentproteinstaining.AdditionalInformationA.PleasevisitthePiercewebsiteforadditionalinformationincludingthefollowingitem:TechTip:Attachanantibodyontoglass,silicaorquartzsurfaceB.Two-stepreactionschemeMaleimide-activatedAntibodyAntibody-enzymeConjugateSulfo-SMCCAntibodyAntibodyAntibodyEnzymeEnzymeFigure1.Two-stepreactionschemeforconjugatingantibodyandenzymeproteinswithSulfo-SMCC.Inthisexample,thecrosslinkerisfirstreactedwiththeantibodytoproduceamal