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

2、。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 SMC。C 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 (succinimidyl 4-N-maleimidomethylcyclohexane-1-carboxylate), 50 mgMolecular We

3、ight:Spacer Arm:Net Mass Added:Product isStorage: Upon receipt store desiccated at 4 C.shipped at ambient temperature.Sulfo-SMCC (sulfosuccinimidyl 4-N-maleimidomethylcyclohexan e-1-carboxylate), 1 g Sulfo-SMCC, 50 mgSulfo-SMCC, No-Weigh? Format, 8 2 mg microtubesMolecular Weight: Spacer Arm: ?Net M

4、ass Added: CAS #: 92921-24-9Storage: Upon receipt store desiccated at -20 C. Produc t is shipped at ambient temperature.IntroductionSMCC and its water-soluble analog Sulfo-SMCC are heterobif unctional crosslinkers that contain N-hydroxysuccinimide (NHS) ester and maleimide groups that allow covalent

5、 conjugation ofamine- and sulfhydryl-containingmolecules.NHS estersreactwith primaryaminesatpH 7-9to formamidebonds,whilemaleimides react withsulfhydrylgroupsat pHtoform stable thioetherbonds.Inaqueoussolutions,NHSesterhydrolytic degradation is a competing reaction whose rate increases with pH. The

6、maleimide group is more stable than the NHS- ester group but will slowly hydrolyze and loses its reaction specificityfor sulfhydrylsat pH values . For these reare usually perforasons, conjugations with these crosslinkers med at pH with the NHS-ester (amine-targeted) reacted befor e or simultaneous w

7、ith the maleimide (sulfhydryl-targeted) rea ction.The cyclohexane ring in the spacer arm of these reagents decreases the rate of hydrolysis of the maleimide group co mpared to similar reagents that do not contain this This feature enables proteins that have been maleimide-activated wi th SMCC or Sul

8、fo-SMCC to be lyophilized and stored for late r conjugation to a sulfhydryl-containing molecule. Many maleim ide-activated protein products are produced in this manner (s ee Related Products).SMCC and Sulfo-SMCC are often used to prepare antibody-en zyme and hapten-carrier protein conjugates in a tw

9、o-step reac tion scheme. First, the amine-containing protein is reacted w ith a several-fold molar excess of the crosslinker, followed by removal of excess (nonreacted) reagent by desalting or dialysis; finally, the sulfhydryl-containing molecule is added to react with the maleimide groups already a

10、ttached to the first protein.Sulfo-SMCC is soluble in water and many other aqueous bu ffers to approximately 10 mM, although solubility decreases w ith increasing salt concentration. SMCC is not directly water -soluble and must be dissolved in an organic solvent such a s dimethylsulfoxide (DMSO) or

11、dimethylformamide (DMF); subseque nt dilution into aqueous reaction buffer is generally possibl e, and most protein reactants will remain soluble if the fi nal concentration of organic solvent is less than 10%.SMCC and Sulfo-SMCCImportantProduct InformationSMCC andSulfo-SMCC aremoisture-sensitive.St

12、ore reagentvial in desiccant. Equilibratevial to room temperature before opening to avoid moisturecondensation inside the containerDissolve needed amount ofreareagent and use it immediately before hydrolysis occurs. Discard any unused reconstitutedNo-Weigh MicrotubeHandling:Immediatelybeforeuse,punc

13、ture the microtubefoilwith apipette tip,add200lof50 mM sodium phosphatebuffer(pH or ultrapurewaterandpipette up anddown to mix.After use,cut theusedmicrotube from the microtubestripand discard.Storetheunusedmicrotubes in the foilpouchprovided.Note: Do notusephosphate-buffered saline (PBS)forinitial

14、dissolutionof Sulfo-SMCC;the reagentdoesnotdissolvewell in buffersexceeding 50 mM totalsalts.However,oncedissolved, thesolution canbe furtherdilutedinPBS orother non-aminebuffers.?solution.gent. Do not store reagent inAvoidbufferscontaining primaryamines .,Tris orglycine)andsulfhydrylsduring conjuga

15、tion,becausethey willcompetewiththe intended reaction. Ifnecessary,dialyzeordesaltsamples intoan appropriate buffer suchas phosphate-buffered saline (PBS).Molecules to be reacted with the maleimide moiety must h ave free (reduced) sulfhydryls. Reduce peptide disulfide bonds with Immobilized TCEP Dis

16、ulfide Reducing Gel (Product No. 7 7712). For proteins, reduce disulfide bonds using 5 mM TCEPir(1:100 dilution77720) forfollowedZeba?antibodies)disulfideidebonds inof Bond-Breaker ? TCEP Solution,30 minutes at roomtemperature,Product No.by twoDesaltmay bebonds.passes throughSpin Columns).a suitable

17、Be awareinactivated by completedesalting columthat proteinsreduction of theSelective reduction of hinge-region disulfIgG can be accomplished with 2-Mercaptoethylamine? HCl (2-MEA, Product No. 20408). Sulfhydryls can be added to molecules using N-succinimidyl S-acetylthioacetate (SATA, Pr oduct No. 2

18、6102) or 2-iminothiolane ? HCl (Traut s Reagent, Pr oduct No. 26101), which modify primary amines.Procedure forTwo-step ProteinCrosslinkingGenerally,a 10- to 50-fold molar excess of crosslinkerover theamount of amine-containingprotein results in sufficngproteinstobe conjugated toeachamine-containing

19、protein.More diluteprotein solutionsrequiregreater foldmolar excess of reagent to achieve thesameactivation level.Empiricaltestingisnecessary to determineoptimal activation levelsand finalconjugation ratiosforthe intended applicationient maleimide activationto enable several sulfhydryl-containiA. Ma

20、terial PreparationConjugation Buffer: phosphate-buffered saline (PBS = 100 m M sodium phosphate, 150 mM sodium chloride, pH ; ., Product No. 28372) or other amine- and sulfhydryl-free buffer at p H (see Important Product Information) adding EDTA to 15 mM helps to chelate divalent metals, thereby red

21、ucing disu lfide formation in the sulfhydryl-containing protein? Desalting column to separate modified protein from exc ess crosslinker and reaction byproducts ., Zeba Desalt Spin Columns)?Amine-containing (Protein-NH2) and sulfhydryl-containing prot eins (Protein-SH) to be conjugatedB. ProtocolNote

22、: For best results, ensure that Protein-SH is prepare d and ready to combine with Protein-NH2 in step 5. 1. Prep are Protein-NH2 in Conjugation Buffer.2. Add the appropriate amount of crosslinker to the prot ein solution. The concentration of the Protein-NH2 determines thecrosslinker molar excess to

23、 use. Suggested crosslinker mol ar excesses are as follows (also see Table 1):? Protein samples 1 mg/ml use 40-80-fold molar excess.? Protein samples of 1-4 mg/ml use 20-fold molar excess? Protein samples of 5-10 mg/ml use 5- to 10-fold mola r excess.Table 1. Crosslinker preparation and molar excess

24、 to usefor1 ml of sample.Immediatelybeforeuse, dissolvecrosslinkerin the appropriatesolventat theconcentrationdenoted inparentheses; thenadd thelisted volume to a 1ml proteinsample. For example,to use the No-Weigh Sulfo-SMCC, dissolvethe2 mg contentsof themicrotubein 200l ofbufferandthen add the pre

25、scribedvolumeto per 1ml sample. For theother products,theappropriateamountof dry reagentmustbe weighed ona balance .,mg Sulfo-SMCC fordissolution in 500 l buffer).Protein-NH2 Concentration(based on a 50 kDa protein) 10 mg/ml 1 mg/ml mg /mlCrosslinker Molar Excess 5X 20X 50X Sulfo-SMCC(in 50 mM sodiu

26、m phosphate or water) 100 lmg/ml*)40 lmg/ml*) 50 lmg/ml*) No-Weigh Sulfo-SMCC(in 50 mM sodium phosphate or water)50 l (10 mg/ml*) 20 l(10 mg/ml*) 25 l (10 mg/ml*) SMCC(in DMSO or DMF)100 l mg/ml*)100 l mg/ml*)100 l mg/ml*)*Concentration of each crosslinker before adding to protei n sample.Note: If t

27、he Sulfo-SMCC solution does not completely diss olve, place the tube under hot running water or incubate fo r several minutes in a 50C water bath.3. Incubate reaction mixture for 30 minutes at room temp erature or 2 hours at 4C.4. Remove excess crosslinker using a desalting column equ ilibrated with

28、 Conjugation Buffer.5. Combine and mix Protein-SH and desalted Protein-NH2 in a molar ratio corresponding to that desired for the finalconjugate and consistent with the relative number of sulf hydryl and activated amines that exist on the two proteins.6. Incubate the reaction mixture at room tempera

29、ture for 3 0 minutes or 2 hours at 4 C.Note: Generally, there is no harm in allowing the reacti on to proceed for several hours or overnight, although usual ly the reaction will be complete in the specified time. To stop the conjugation reaction before completion, add buffer containing reduced cyste

30、ine at a concentration several times greater than the sulfhydryls of Protein-SH. Note: Conjugatio n efficiency can be estimated by electrophoresis separation a nd subsequent protein staining.Additional InformationA. Please visit the Pierce website for additional informa tion including the following

31、item: ?Tech Tip: Attach an antibody onto glass, silicaor quart z surface B. Two-step reaction schemeMaleimide-activated AntibodyAntibody-enzyme ConjugateSulfo-SMCCAntibodyAntibodyAntibodyEnzymeEnzymeFigure 1. Two-stepreaction scheme for conjugating antibody and enzyme proteinswith Sulfo-SMCC. In thi

32、s example, thecrosslinker is firstreacted with the antibody to producea maleimide-activatedprotein. After excess non-reacted crosslinker and by-productsare removed, the maleimide-activatedantibody is reacted withthe appropriate molar ratio of enzymehaving sulfhydrylgroups. Usually,severalor multiple

33、 maleimide-activationsoccur per antibodymolecule,enabling several enzyme molecules to be conjugatedto eachantibody molecule.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（ 马来酰亚胺基团一端 ） 可与另一蛋白质分子的巯基交联。NHS活性酯与伯胺在 PH7-9 的环境形成酰胺键。马来酰亚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。马来酰亚 胺比 NHS稳定，但是在 PH大于时，马来酰亚胺会慢慢水解，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亚胺的水解速率。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