SMCC-结构反应解释

1、.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。很多蛋白质都选用该试剂

2、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精品.smcc(succinimidyl4-n-maleimidomethylcyclohexane-1-carboxylate),50mgmolecularweight:3

3、34.32spacerarm:8.3netmassadded:219.09storage:uponreceiptstoredesiccatedat4c.productisshippedatambienttemperature.sulfo-smcc(sulfosuccinimidyl4-n-maleimidomethylcyclohexane-1-carboxylate),1gsulfo-smcc,50mgsulfo-smcc,no-weighformat,82mgmicrotubesmolecularweight:436.37spacerarm:8.3netmassadded:219.09ca

4、s#:92921-24-9storage:uponreceiptstoredesiccatedat-20c.productisshippedatambienttemperature.introductionsmccanditswater-solubleanalogsulfo-smccareheterobifunctionalcrosslinkersthatcontainn-hydroxysuccinimide(nhs)esterandmaleimidegroupsthatallowcovalentconjugationofamine-andsulfhydryl-containingmolecu

5、les.nhsestersreactwithprimaryaminesatph7-9toformamidebonds,whilemaleimidesreactwithsulfhydrylgroupsatph6.5-7.5toformstablethioetherbonds.inaqueoussolutions,nhsesterhydrolyticdegradationisacompetingreactionwhoserateincreaseswithph.themaleimidegroupismorestablethanthenhs-estergroupbutwillslowlyhydroly

6、zeandlosesitsreactionspecificityforsulfhydrylsatphvalues7.5.forthesereasons,conjugationswiththesecrosslinkersareusuallyperformedatph7.2-7.5,withthenhs-ester(amine-targeted)reactedbeforeorsimultaneouswiththemaleimide(sulfhydryl-targeted)reaction.精品.thecyclohexaneringinthespacerarmofthesereagentsdecre

7、asestherateofhydrolysisofthemaleimidegroupcomparedtosimilarreagentsthatdonotcontainthisring.1thisfeatureenablesproteinsthathavebeenmaleimide-activatedwithsmccorsulfo-smcctobelyophilizedandstoredforlaterconjugationtoasulfhydryl-containingmolecule.manymaleimide-activatedproteinproductsareproducedinthi

8、smanner(seerelatedproducts).smccandsulfo-smccareoftenusedtoprepareantibody-enzymeandhapten-carrierproteinconjugatesinatwo-stepreactionscheme.first,theamine-containingproteinisreactedwithaseveral-foldmolarexcessofthecrosslinker,followedbyremovalofexcess(nonreacted)reagentbydesaltingordialysis;finally

9、,thesulfhydryl-containingmoleculeisaddedtoreactwiththemaleimidegroupsalreadyattachedtothefirstprotein.sulfo-smccissolubleinwaterandmanyotheraqueousbufferstoapproximately10mm,althoughsolubilitydecreaseswithincreasingsaltconcentration.smccisnotdirectlywater-solubleandmustbedissolvedinanorganicsolvents

10、uchasdimethylsulfoxide精品.(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.精品.avoidbufferscontainingprimaryamines(e.g.,trisorglycine)andsulfhydrylsduringconjugation,becausetheywillcompetewiththeintendedreaction.ifnecessary,dia

14、lyzeordesaltsamplesintoanappropriatebuffersuchasphosphate-bufferedsaline(pbs).moleculestobereactedwiththemaleimidemoietymusthavefree(reduced)sulfhydryls.reducepeptidedisulfidebondswithimmobilizedtcepdisulfidereducinggel(productno.77712).forproteins,reducedisulfidebondsusing5mmtcep(1:100dilutionofbon

15、d-breakertcepsolution,productno.77720)for30minutesatroomtemperature,followedbytwopassesthroughasuitabledesaltingcolumn(e.g.,zebadesaltspincolumns).beawarethatproteins(e.g.,antibodies)maybeinactivatedbycompletereductionoftheirdisulfidebonds.selectivereductionofhinge-regiondisulfidebondsiniggcanbeacco

16、mplishedwith2-mercaptoethylaminehcl(2-mea,productno.20408).sulfhydrylscanbeaddedtomoleculesusingn-succinimidyls-acetylthioacetate(sata,productno.26102)or2-iminothiolanehcl(trautsreagent,productno.26101),whichmodifyprimaryamines.procedurefortwo-stepproteincrosslinkinggenerally,a10-to50-foldmolarexces

17、sofcrosslinkerovertheamountofamine-containingproteinresultsinsufficientmaleimideactivationtoenableseveralsulfhydryl-containingproteinstobeconjugatedtoeachamine-containingprotein.morediluteproteinsolutionsrequiregreaterfoldmolarexcessofreagenttoachievethesameactivationlevel.empiricaltestingisnecessar

18、ytodetermineoptimalactivationlevelsandfinalconjugationratiosfortheintendedapplication.a.materialpreparation精品.conjugationbuffer:phosphate-bufferedsaline(pbs=100mmsodiumphosphate,150mmsodiumchloride,ph7.2;e.g.,productno.28372)orotheramine-andsulfhydryl-freebufferatph6.5-7.5(seeimportantproductinforma

19、tion)addingedtato1-5mmhelpstochelatedivalentmetals,therebyreducingdisulfideformationinthesulfhydryl-containingproteindesaltingcolumntoseparatemodifiedproteinfromexcesscrosslinkerandreactionbyproducts(e.g.,zebadesaltspincolumns)amine-containing(protein-nh2)andsulfhydryl-containingproteins(protein-sh)tobeconjugatedb.protocolnote:forbestresults,ensurethatprotein-shispreparedandreadytocombi