SMCC-結(jié)構(gòu)反應(yīng)解釋

8.3A 8.3AN-0SMCC是一類含有N-羥基琥珀酰亞胺(NHS)活性酯和馬來(lái)酰亞胺的雙功能偶聯(lián)劑.可以將分別含有巰基和氨基的化合物鍵接在一起。NHS活性酯與伯胺在PH7-9的環(huán)境形成酰胺鍵。馬來(lái)酰胺與巰基在PH6.5-7.5的環(huán)境下形成穩(wěn)定的硫醚鍵。在水溶液中，NHS活性酯的水解是(與氨基的反應(yīng))個(gè)競(jìng)爭(zhēng)反應(yīng)。馬來(lái)酰胺比NHS穩(wěn)定，但是在PH大于7.5時(shí)，馬來(lái)酰胺會(huì)慢慢水解，失去與巰基反應(yīng)的特異性。因而，在使用SMCC時(shí)通常是在PH7.2-7.5的環(huán)境下進(jìn)行，并且先讓NHS發(fā)生反應(yīng)。SMCC結(jié)構(gòu)里的環(huán)己烷環(huán)可以降低馬來(lái)酰胺的水解速率。這使得蛋白質(zhì)在用SMCC修飾之后可以凍干存放一段時(shí)間。很多蛋白質(zhì)都選用該試劑來(lái)進(jìn)行馬來(lái)酰亞胺修飾。用SMCC來(lái)制備抗體-酶或者半抗原作載體的蛋白質(zhì)，經(jīng)常采用兩步合成法。首先，含有氨基的蛋白質(zhì)與幾倍的偶聯(lián)劑反應(yīng)，反應(yīng)結(jié)束后通過(guò)脫鹽柱或者透析的方法除掉沒(méi)有反應(yīng)玩的SMCC。然后，再與含有巰基的蛋白質(zhì)反應(yīng)。在實(shí)際操作中要注意的是，SMCC怕潮濕，存放時(shí)要和干燥劑一起存放。并且使用中從冰箱拿出來(lái)時(shí)要先在室外放置一段時(shí)間平衡溫度，以免立刻開啟，空氣中水分遇冷凝結(jié)，破壞SMCC結(jié)構(gòu)。INSTRUCTIONSSMCC(succinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate),50mgMolecularWeight:334.32SpacerArm:8.3ANetMassAdded:219.09Storage:Uponreceiptstoredesiccatedat4°C.Productisshippedatambienttemperature.Sulfo-SMCC(sulfosuccinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate),1gSulfo-SMCC,50mgSulfo-SMCC,No-Weigh?Format,8*2mgmicrotubesMolecularWeight:436.37SpacerArm:8.3ANetMassAdded:219.09CAS#:92921249Storage:Uponreceiptstoredesiccatedat20°C.Productisshippedatambienttemperature.IntroductionSMCCanditswatersolubleanalogSulfoSMCCareheterobifunctionalcrosslinkersthatcontainNhydroxysuccinimide(NHS)esterandmaleimidegroupsthatallowcovalentconjugationofamineandsulfhydrylcontainingmolecules.NHSestersreactwithprimaryaminesatpH79toformamidebonds,whilemaleimidesreactwithsulfhydrylgroupsatpH6.57.5toformstablethioetherbonds.Inaqueoussolutions,NHSesterhydrolyticdegradationisacompetingreactionwhoserateincreaseswithpH.ThemaleimidegroupismorestablethantheNHSestergroupbutwillslowlyhydrolyzeandlosesitsreactionspecificityforsulfhydrylsatpHvalues>7.5.Forthesereasons,conjugationswiththesecrosslinkersareusuallyperformedatpH7.27.5,withtheNHSester(aminetargeted)reactedbeforeorsimultaneouswiththemaleimide(sulfhydryltargeted)reaction.Thecyclohexaneringinthespacerarmofthesereagentsdecreasestherateofhydrolysisofthemaleimidegroupcomparedtosimilarreagentsthatdonotcontainthisring.1ThisfeatureenablesproteinsthathavebeenmaleimideactivatedwithSMCCorSulfoSMCCtobelyophilizedandstoredforlaterconjugationtoasulfhydrylcontainingmolecule.Manymaleimideactivatedproteinproductsareproducedinthismanner(seeRelatedProducts).SMCCandSulfoSMCCareoftenusedtoprepareantibodyenzymeandhaptencarrierproteinconjugatesinatwostepreactionscheme.First,theaminecontainingproteinisreactedwithaseveralfoldmolarexcessofthecrosslinker,followedbyremovalofexcess(nonreacted)reagentbydesaltingordialysisfinally,thesulfhydrylcontainingmoleculeisaddedtoreactwiththemaleimidegroupsalreadyattachedtothefirstprotein.SulfoSMCCissolubleinwaterandmanyotheraqueousbufferstoapproximately10mM,althoughsolubilitydecreaseswithincreasingsaltconcentration.SMCCisnotdirectlywatersolubleandmustbedissolvedinanorganicsolventsuchasdimethylsulfoxide(DMSO)ordimethylformamide(DMF)subsequentdilutionintoaqueousreactionbufferisgenerallypossible,andmostproteinreactantswillremainsolubleifthefinalconcentrationoforganicsolventislessthan10%.SMCCandSulfoSMCCImportantProductInformationSMCCandSulfoSMCCaremoisturesensitive.Storereagentvialindesiccant.Equilibratevialtoroomtemperaturebeforeopeningtoavoidmoisturecondensationinsidethecontainer.Dissolveneededamountofreagentanduseitimmediatelybeforehydrolysisoccurs.Discardanyunusedreconstitutedreagent.Donotstorereagentinsolution.?NoWeighMicrotubeHandling:Immediatelybeforeuse,puncturethemicrotubefoilwithapipettetip,add200^lof50mMsodiumphosphatebuffer(pH7.0orultrapurewaterandpipetteupanddowntomix.Afteruse,cuttheusedmicrotubefromthemicrotubestripanddiscard.Storetheunusedmicrotubesinthefoilpouchprovided.Note:Donotusephosphatebufferedsaline(PBS)forinitialdissolutionofSulfoSMCCthereagentdoesnotdissolvewellinbuffersexceeding50mMtotalsalts.However,oncedissolved,thesolutioncanbefurtherdilutedinPBSorothernonaminebuffers.?Avoidbufferscontainingprimaryamines(e.g.,Trisorglycine)andsulfhydrylsduringconjugation,becausetheywillcompetewiththeintendedreaction.Ifnecessary,dialyzeordesaltsamplesintoanappropriatebuffersuchasphosphatebufferedsaline(PBS).?Moleculestobereactedwiththemaleimidemoietymusthavefree(reduced)sulfhydryls.ReducepeptidedisulfidebondswithImmobilizedTCEPDisulfideReducingGel(ProductNo.77712).Forproteins,reducedisulfidebondsusing5mMTCEP(1:100dilutionofBondBreaker?TCEPSolution,ProductNo.77720)for30minutesatroomtemperature,followedbytwopassesthroughasuitabledesaltingcolumn(e.g.,Zeba?DesaltSpinColumns).Beawarethatproteins(e.g.,antibodies)maybeinactivatedbycompletereductionoftheirdisulfidebonds.SelectivereductionofhingeregiondisulfidebondsinIgGcanbeaccomplishedwith2Mercaptoethylamine?HCl(2MEA,ProductNo.20408).SulfhydrylscanbeaddedtomoleculesusingNsuccinimidylSacetylthioacetate(SATA,ProductNo.26102)or2iminothiolane?HCl(Traut,sReagent,ProductNo.26101),whichmodifyprimaryamines.ProcedureforTwostepProteinCrosslinkingGenerally,a10to50foldmolarexcessofcrosslinkerovertheamountofaminecontainingproteinresultsinsufficientmaleimideactivationtoenableseveralsulfhydrylcontainingproteinstobeconjugatedtoeachaminecontainingprotein.Morediluteproteinsolutionsrequiregreaterfoldmolarexcessofreagenttoachievethesameactivationlevel.Empiricaltestingisnecessarytodetermineoptimalactivationlevelsandfinalconjugationratiosfortheintendedapplication.A.MaterialPreparation?ConjugationBuffer:phosphatebufferedsaline(PBS=100mMsodiumphosphate,150mMsodiumchloride,pH7.2e.g.,ProductNo.28372)orotheramineandsulfhydrylfreebufferatpH6.5.5(seeImportantProductInformation)—addingEDTAto1mMhelpstochelatedivalentmetals,therebyreducingdisulfideformationinthesulfhydrylcontainingprotein?Desaltingcolumntoseparatemodifiedproteinfromexcesscrosslinkerandreactionbyproducts(e.g.,ZebaDesaltSpinColumns)?Aminecontaining(ProteinNH2)andsulfhydrylcontainingproteins(ProteinSH)tobeconjugatedB.ProtocolNote:Forbestresults,ensurethatProteinSHispreparedandreadytocombinewithProteinNH2instep5.1.PrepareProteinNH2inConjugationBuffer.Addtheappropriateamountofcrosslinkertotheproteinsolution.TheconcentrationoftheProteinNH2determinesthecrosslinkermolarexcesstouse.Suggestedcrosslinkermolarexcessesareasfollows(alsoseeTable1):Proteinsamples<1mg/mluse4080foldmolarexcess.Proteinsamplesof14mg/mluse20foldmolarexcess.Proteinsamplesof510mg/mluse5to10foldmolarexcess.Table1.Crosslinkerpreparationandmolarexcesstousefor1mlofsample.Immediatelybeforeuse,dissolvecrosslinkerintheappropriatesolventattheconcentrationdenotedinparenthesesthenaddthelistedvolumetoa1mlproteinsample.Forexample,tousetheNoWeighSulfoSMCC,dissolvethe2mgcontentsofthemicrotubein200^lofbufferandthenaddtheprescribedvolumetoper1mlsample.Fortheotherproducts,theappropriateamountofdryreagentmustbeweighedonabalance(e.g.,2.4mgSulfoSMCCfordissolutionin500^lbuffer).ProteinNH2Concentration(basedona50kDaprotein)10mg/ml1mg/ml0.5mg/mlCrosslinkerMolarExcess5X20X50XSulfoSMCC(in50mMsodiumphosphateorwater)100^l(4.8mg/ml*)40^l(4.8mg/ml*)50^1(4.8mg/ml*)NoWeighSulfoSMCC(in50mMsodiumphosphateorwater)50^1(10mg/ml*)20^1(10mg/ml*)25^1(10mg/ml*)SMCC(inDMSOorDMF)100^l(3.7mg/ml*)100^l(1.5mg/ml*)100^l(1.8mg/ml*)"Concentrationofeachcrosslinkerbeforeaddingtoproteinsample.Note:IftheSulfoSMCCsolutiondoesnotcompletelydissolve,placethetubeunderhotrunningwaterorincubateforseveralminutesina50°Cwaterbath.Incubatereactionmixturefor30minutesatroomtemperatureor2hoursat4°C.RemoveexcesscrosslinkerusingadesaltingcolumnequilibratedwithConjugationBuffer.CombineandmixProteinSHanddesaltedProteinNH2inamolarratiocorrespondingtothatdesiredforthefinalconjugateandconsistentwiththerelativenumberofsulfhydrylandactivatedaminesthatexistonthetwoproteins.6.Incubatethereactionmixtureatroomtemperaturefor30minutesor2hoursat4°C.Note:Generally,thereisnoharminallowingthereactiontoproceedforseveralhoursorovernight,althoughusuallythereactionwillbecompleteinthespecifiedtime.Tostoptheconjugationreactionbeforecompletion,addbuffercontainingreducedcysteineataconcentrationseveraltimesgreaterthanthesulfhydrylsofProteinSH.Note:Conjugationefficiencycanbeestimatedbyelectrophoresisseparationandsubsequentproteinstaining.AdditionalInformationA.PleasevisitthePiercewebsiteforadditionalinformationincludingthefollowingitem:?TechTip:Attachanantibodyontoglass,silicaorquartzsurfaceB.TwostepreactionschemeMaleimideactivatedAntibodyAntibodyenzymeConjugateSulfoSMCCAntibodyAntibodyAntibodyEnzymeEnzymeFigure1.TwostepreactionschemeforconjugatingantibodyandenzymeproteinswithSulfoSMCC.Inthisexample,thecrosslinkerisfirstreactedwiththeantibodytoproduceamaleimideactivatedprotein.Afterexcessnonreactedcrosslinkerandbyproductsareremoved,themaleimideactivatedantibodyisreactedwiththeappropriatemolarratioofenzymehavingsulfhydrylgroups.Usually,severalormultiplemaleimideactivationsoccurperantibodymolecule,enablingseveralenzymemoleculestobeconjugatedtoeachantibodymolecule.MBS/BDB/SMCC/sulfoSMCC1、SMCC琥珀酰亞胺4（N馬來(lái)酰亞胺）環(huán)已烷11羥酸酯分子一端的NHS酯基團(tuán)與某一蛋白質(zhì)分子的伯氨反應(yīng)形成穩(wěn)定的酰胺鍵，另一端（馬來(lái)酰亞胺基團(tuán)一端）可與另一蛋白質(zhì)分子的巰基交聯(lián)。NHS活性酯與伯胺在PH79的環(huán)境形成酰