SMCC_結(jié)構(gòu)反應(yīng)解釋

1、SMCC是一類含有N-羥基琥珀酰亞胺(NHS)活性酯和馬來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))個競爭反應(yīng)。馬來酰胺比NHS穩(wěn)定，但是在PH大于7.5時，馬來酰胺會慢慢水解，失去與巰基反應(yīng)的特異性。因而，在使用SMCC時通常是在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間。很多蛋白質(zhì)都選用該試劑來進(jìn)

2、行馬來酰亞胺修飾。用SMCC來制備抗體-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)束后通過脫鹽柱或者透析的方法除掉沒有反應(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，存放時要和干燥劑一起存放。并且使用中從冰箱拿出來時要先在室外放置一段時間平衡溫度，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-maleimidomethylcyclohexane-1-carboxylate),50mgMolecularWeight:334.32S

3、pacerArm:8.3ANetMassAdded:219.09Storage:Uponreceiptstoredesiccatedat4C.Productisshippedatambienttemperature.Sulfo-SMCC(sulfosuccinimidyl4-N-maleimidomethylcyclohexane-1-carboxylate),1gSulfo-SMCC,50mgSulfo-SMCC,No-WeighFormat,8x2mgmicrotubesMolecularWeight:436.37SpacerArm:8.3ANetMassAdded:219.09CAS#:

4、92921249Storage:Uponreceiptstoredesiccatedat20C.Productisshippedatambienttemperature.IntroductionSMCCanditswatersolubleanalogSulfoSMCCareheterobifunctionalcrosslinkersthatcontainNhydroxysuccinimide(NHS)esterandmaleimidegroupsthatallowcovalentconjugationofamineandsulfhydrylcontainingmolecules.NHSeste

5、rsreactwithprimaryaminesatpH79toformamidebonds,whilemaleimidesreactwithsulfhydrylgroupsatpH6.57.5toformstablethioetherbonds.Inaqueoussolutions,NHSesterhydrolyticdegradationisacompetingreactionwhoserateincreaseswithpH.ThemaleimidegroupismorestablethantheNHSestergroupbutwillslowlyhydrolyzeandlosesitsr

6、eactionspecificityforsulfhydrylsatpHvalues7.5.Forthesereasons,conjugationswiththesecrosslinkersareusuallyperformedatpH7.27.5,withtheNHSester(aminetargeted)reactedbeforeorsimultaneouswiththemaleimide(sulfhydryltargeted)reaction.Thecyclohexaneringinthespacerarmofthesereagentsdecreasestherateofhydrolys

7、isofthemaleimidegroupcomparedtosimilarreagentsthatdonotcontainthisring.1ThisfeatureenablesproteinsthathavebeenmaleimideactivatedwithSMCCorSulfoSMCCtobelyophilizedandstoredforlaterconjugationtoasulfhydrylcontainingmolecule.Manymaleimideactivatedproteinproductsareproducedinthismanner(seeRelatedProduct

8、s).SMCCandSulfoSMCCareoftenusedtoprepareantibodyenzymeandhaptencarrierproteinconjugatesinatwostepreactionscheme.First,theaminecontainingproteinisreactedwithaseveralfoldmolarexcessofthecrosslinker,followedbyremovalofexcess(nonreacted)reagentbydesaltingordialysisfinally,thesulfhydrylcontainingmolecule

9、isaddedtoreactwiththemaleimidegroupsalreadyattachedtothefirstprotein.SulfoSMCCissolubleinwaterandmanyotheraqueousbufferstoapproximately10mM,althoughsolubilitydecreaseswithincreasingsaltconcentration.SMCCisnotdirectlywatersolubleandmustbedissolvedinanorganicsolventsuchasdimethylsulfoxide(DMSO)ordimet

10、hylformamide(DMF)subsequentdilutionintoaqueousreactionbufferisgenerallypossible,andmostproteinreactantswillremainsolubleifthefinalconcentrationoforganicsolventislessthan10%.SMCCandSulfoSMCCImportantProductInformationSMCCandSulfoSMCCaremoisturesensitive.Storereagentvialindesiccant.Equilibratevialtoro

11、omtemperaturebeforeopeningtoavoidmoisturecondensationinsidethecontainer.Dissolveneededamountofreagentanduseitimmediatelybeforehydrolysisoccurs.Discardanyunusedreconstitutedreagent.Donotstorereagentinsolution.NoWeighMicrotubeHandling:Immediatelybeforeuse,puncturethemicrotubefoilwithapipettetip,add200

12、卩1of50mMsodiumphosphatebuffer(pH7.07.5)orultrapurewaterandpipetteupanddowntomix.Afteruse,cuttheusedmicrotubefromthemicrotubestripanddiscard.Storetheunusedmicrotubesinthefoi1pouchprovided.Note:Donotusephosphatebufferedsa1ine(PBS)forinitia1disso1utionofSu1foSMCCthereagentdoesnotdisso1vewe11inbuffersex

13、ceeding50mMtota1sa1ts.However,oncedisso1ved,theso1utioncanbefurtherdi1utedinPBSorothernonaminebuffers.Avoidbufferscontainingprimaryamines(e.g.,Trisorg1ycine)andsu1fhydry1sduringconjugation,becausetheywi11competewiththeintendedreaction.Ifnecessary,dia1yzeordesa1tsamp1esintoanappropriatebuffersuchasph

14、osphatebufferedsa1ine(PBS).Mo1ecu1estobereactedwiththema1eimidemoietymusthavefree(reduced)su1fhydry1s.Reducepeptidedisu1fidebondswithImmobi1izedTCEPDisu1fideReducingGe1(ProductNo.77712).Forproteins,reducedisu1fidebondsusing5mMTCEP(1:100di1utionofBondBreakerTCEPSo1ution,ProductNo.77720)for30minutesat

15、roomtemperature,fo11owedbytwopassesthroughasuitab1edesa1tingco1umn(e.g.,ZebaDesa1tSpinCo1umns).Beawarethatproteins(e.g.,antibodies)maybeinactivatedbycomp1etereductionoftheirdisu1fidebonds.Se1ectivereductionofhingeregiondisu1fidebondsinIgGcanbeaccomp1ishedwith2MercaptoethylamineHCl(2MEA,ProductNo.204

16、08).Su1fhydry1scanbeaddedtomo1ecu1esusingNsuccinimidylSacetylthioacetate(SATA,ProductNo.26102)or2iminothiolaneHCl(TrautsReagent,ProductNo.26101),whichmodifyprimaryamines.ProcedureforTwostepProteinCrosslinkingGenerally,a10to50foldmolarexcessofcrosslinkerovertheamountofaminecontainingproteinresultsins

17、ufficientmaleimideactivationtoenableseveralsulfhydrylcontainingproteinstobeconjugatedtoeachaminecontainingprotein.Morediluteproteinsolutionsrequiregreaterfoldmolarexcessofreagenttoachievethesameactivationlevel.Empiricaltestingisnecessarytodetermineoptimalactivationlevelsandfinalconjugationratiosfort

18、heintendedapplication.A.MaterialPreparationConjugationBuffer:phosphatebufferedsaline(PBS=100mMsodiumphosphate,150mMsodiumchloride,pH7.2e.g.,ProductNo.28372)orotheramineandsulfhydrylfreebufferatpH6.57.5(seeImportantProductInformation)addingEDTAto15mMhelpstochelatedivalentmetals,therebyreducingdisulfi

19、deformationinthesulfhydrylcontainingproteinDesaltingcolumntoseparatemodifiedproteinfromexcesscrosslinkerandreactionbyproducts(e.g.,ZebaDesaltSpinColumns)Aminecontaining(ProteinNH2)andsulfhydrylcontainingproteins(ProteinSH)tobeconjugatedB.ProtocolNote:Forbestresults,ensurethatProteinSHispreparedandre

20、adytocombinewithProteinNH2instep5.1.PrepareProteinNH2inConjugationBuffer.Addtheappropriateamountofcrosslinkertotheproteinsolution.TheconcentrationoftheProteinNH2determinesthecrosslinkermolarexcesstouse.Suggestedcrosslinkermolarexcessesareasfollows(alsoseeTable1):Proteinsamples1mg/mluse4080foldmolare

21、xcess.Proteinsamplesof14mg/mluse20foldmolarexcess.Proteinsamplesof510mg/mluse5to10foldmolarexcess.Table1.Crosslinkerpreparationandmolarexcesstousefor1mlofsample.Immediatelybeforeuse,dissolvecrosslinkerintheappropriatesolventattheconcentrationdenotedinparenthesesthenaddthelistedvolumetoa1mlproteinsam

22、ple.Forexample,tousetheNoWeighSulfoSMCC,dissolvethe2mgcontentsofthemicrotubein200卩1ofbufferandthenaddtheprescribedvolumetoper1mlsample.Fortheotherproducts,theappropriateamountofdryreagentmustbeweighedonabalance(e.g.,2.4mgSulfoSMCCfordissolutionin500卩1buffer).ProteinNH2Concentration(basedona50kDaprot

23、ein)10mg/ml1mg/ml0.5mg/mlCrosslinkerMolarExcess5X20X50XSulfoSMCC(in50mMsodiumphosphateorwater)100卩1(4.8mg/ml*)40卩1(4.8mg/ml*)50卩1(4.8mg/ml*)NoWeighSulfoSMCC(in50mMsodiumphosphateorwater)50卩1(10mg/ml*)20卩1(10mg/ml*)25卩1(10mg/ml*)SMCC(inDMSOorDMF)100卩l(xiāng)(3.7mg/ml*)100卩l(xiāng)(1.5mg/ml*)100卩l(xiāng)(1.8mg/ml*)*Concen

24、trationofeachcrosslinkerbeforeaddingtoproteinsample.Note:IftheSulfoSMCCsolutiondoesnotcompletelydissolve,placethetubeunderhotrunningwaterorincubateforseveralminutesina50Cwaterbath.Incubatereactionmixturefor30minutesatroomtemperatureor2hoursat4C.Removeexcesscrosslinkerusingadesaltingcolumnequilibrate

25、dwithConjugationBuffer.CombineandmixProteinSHanddesaltedProteinNH2inamolarratiocorrespondingtothatdesiredforthefinalconjugateandconsistentwiththerelativenumberofsulfhydrylandactivatedaminesthatexistonthetwoproteins.6.Incubatethereactionmixtureatroomtemperaturefor30minutesor2hoursat4C.Note:Generally,

26、thereisnoharminallowingthereactiontoproceedforseveralhoursorovernight,althoughusuallythereactionwillbecompleteinthespecifiedtime.Tostoptheconjugationreactionbeforecompletion,addbuffercontainingreducedcysteineataconcentrationseveraltimesgreaterthanthesulfhydrylsofProteinSH.Note:Conjugationefficiencyc

27、anbeestimatedbyelectrophoresisseparationandsubsequentproteinstaining.AdditionalInformationA.PleasevisitthePiercewebsiteforadditionalinformationincludingthefollowingitem:TechTip:Attachanantibodyontoglass,silicaorquartzsurfaceB.TwostepreactionschemeMaleimideactivatedAntibodyAntibodyenzymeConjugateSulf

28、oSMCCAntibodyAntibodyAntibodyEnzymeEnzymeFigure1.TwostepreactionschemeforconjugatingantibodyandenzymeproteinswithSulfoSMCC.Inthisexample,thecrosslinkerisfirstreactedwiththeantibodytoproduceamaleimideactivatedprotein.Afterexcessnonreactedcrosslinkerandbyproductsareremoved,themaleimideactivatedantibod

29、yisreactedwiththeappropriatemolarratioofenzymehavingsulfhydrylgroups.Usually,severalormultiplemaleimideactivationsoccurperantibodymolecule,enablingseveralenzymemoleculestobeconjugatedtoeachantibodymolecule.MBS/BDB/SMCC/sulfoSMCC1、SMCC琥珀酰亞胺4（N馬來酰亞胺）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（馬來酰亞胺基團(tuán)

30、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)境形成酰胺鍵。馬來酰亞胺與巰基在PH6.57.5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)）個競爭反應(yīng)。馬來酰亞胺比NHS穩(wěn)定，但是在PH大于7.5時，馬來酰亞胺會慢慢水解，失去與巰基反應(yīng)的特異性。因而，在使用SMCC時通常是在PH7.27.5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)可以降低馬來酰亞胺的水解速率。這使得蛋白質(zhì)在用SMCC修飾之后可以凍干存放一段時間。很多蛋白質(zhì)都選用該試劑來進(jìn)行馬來酰亞胺修飾。用SMCC來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)束后通過脫鹽柱或者透析的方法除掉沒有反應(yīng)完的SMCC。然后，