SMCC-结构反应解释

1、SMCO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.可以将分别含有琉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。马来酰胺与琉基在的环境下形成稳定的硫醴键。在水 溶液中，NHS舌性酯的水解是（与氨基的反应）个竞争反应。马来酰胺比NHSg定，但是在PH大于时，马来酰胺会慢慢水解，失去与琉基反应的特异性。因而， 在使用SMCC通常是在的环境下进行，并且先让 NHS®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SMCO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SMC

2、区制备抗体-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，再与含有琉基的蛋白质反应。在实际操 作中要注意的是，SMCCJ白潮湿，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吉构。INSTRUCTIONSSMCC (succinimidyl 4-N-maleimidomethylcyclohexane-1-carboxylate), 50 mgMolecular Weight:Spacer Arm

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

4、dded: CAS #:92921-24-9Storage: Upon receipt store desiccated at -20°C. Product 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 c

5、onjugation of amine- and sulfhydryl-containingmolecules. NHS estersreactwith primary amines at pH 7-9 to form amide bonds,whilemaleimides react with sulfhydryl groups at pH to form st able thioether bonds. In aqueous solutions, NHS ester hydroly tic degradation is a competing reaction whose rate inc

6、reases withpH. The maleimide group is more stable than the NHS-estergroup but will slowly hydrolyze and loses its reactionspecificity for sulfhydryls at pH values > . For these re asons, conjugations with these crosslinkers are usually perforreacted beformed at pH with the NHS-ester (amine-target

7、ed) e or simultaneous with the maleimide (sulfhydryl-targeted) rea ction.The cyclohexane ring in the spacer arm of these reagents decreases therate ofhydrolysisof the maleimide group compared to similar reagents that do not contain this This feature enablesproteinsthat have been maleimide-activatedw

8、ith SMCC or Sulfo-SMCC to be lyophilized and stored for late r conjugation to a sulfhydryl-containing molecule. Many maleim ide-activatedprotein products areproducedin this manner (see Related Products).SMCC andSulfo-SMCC areoftenused toprepareantibody-enzyme and hapten-carrier protein conjugates in

9、 a two-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 alre

10、ady attached to the first protein.Sulfo-SMCC is soluble in water and many other aqueous bu ffers toapproximately 10 mM, although solubility decreaseswith increasing salt concentration. SMCC is not directly water -solubleand must bedissolved in anorganicsolvent suchas dimethylsulfoxide (DMSO) or dime

11、thylformamide (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-SMCCImportant Product Information ?SMCC and Sulfo-SMCC aremoisture-sensitive.St

12、orereagentvial in desiccant.Equilibrate vial to room temperature before opening to avoid moisture condensation inside the container . Dissolve needed amount ofreagent and use it immediatelybefore hydrolysis occurs. Discard any unused reconstituted rea gent. Do not store reagent insolution.?No-Weigh

13、Microtube Handling:Immediatelybeforeuse,puncture the microtube foil with a pipette tip, add 200 仙 l of 50 mM sodium phosphate buffer(pH orultrapurewaterandpipette up and down to mix.After use,cut theusedmicrotube fromthemicrotubestripand discard.Store the unusedmicrotubesinthe foilpouchprovided.Note

14、: Do not use phosphate-buffered saline (PBS) for init ial dissolution of Sulfo-SMCC; the reagent does not dissolve well in buffers exceeding 50 mM total salts. However, once dissolved, the solution can be further diluted in PBS or other non-amine buffers. ?Avoidbuffers containing primaryamines .,Tri

15、s or glycine) and sulfhydryls during conjugation, because they will comp ete withthe intended reaction. Ifnecessary,dialyzeor desalt samples into an appropriate buffer such as phosphate- bu ffered saline (PBS).Molecules to be reacted with the maleimide moiety must h ave free (reduced) sulfhydryls. R

16、educe peptide disulfide bonds with Immobilized TCEP Disulfide Reducing Gel (Product No. 7 7712). For proteins, reduce disulfide bonds using 5 mM TCEP (1:100 dilution of Bond-Breaker ? TCEP Solution, Product No. 77720) for 30 minutes at room temperature,followedby two passes througha suitable desalti

17、ng column ., Zeba?DesaltSpin Columns).Be aware that proteins .,antibodies) may be inactivated by complete reduction of the irdisulfide bonds. Selective reductionof hinge-region disulfidebonds in IgG can be accomplishedwith2-Mercaptoethylamine? HCl (2-MEA, Product No. 20408). Sulfhydryls can be added

18、 to molecules using N-succinimidyl S-acetylthioacetate (SATA, Pr oductNo. 26102) or 2-iminothiolane ? HCl (Trauts Reagent, ProductNo. 26101), which modify primary amines.Procedure for Two-step Protein CrosslinkingGenerally, a 10- to 50-fold molar excess of crosslinker over the amount of amine-contai

19、ningproteinresults in sufficientmaleimide activation to enableseveralsulfhydryl-containing proteins to be conjugated to each amine-containing protein . More dilute protein solutions require greater fold molar e xcess of reagent to achieve the same activation level. Empir ical testing is necessary to

20、 determine optimal activation lev els and finalconjugationratiosfor the intended application. A. MaterialPreparation?100 mConjugation Buffer: phosphate-buffered saline (PBSM sodium phosphate, 150 mM sodium chloride, pH ;., ProductNo. 28372) or other amine- and sulfhydryl-free buffer at pH (see Impor

21、tant Product Information) - adding EDTA to 15 mM helps to chelate divalent metals, thereby reducing 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

22、 (Protein-NH2) and sulfhydryl-containing prot eins (Protein-SH) to be conjugatedB. ProtocolNote: For best results, ensure that Protein-SH is prepare d and ready to combine with Protein-NH2 in step 5.1. Prepare Protein-NH2 in Conjugation Buffer.2. Add the appropriate amount of crosslinker to the prot

23、 ein solution. The concentration of the Protein-NH2 determines thecrosslinker molar excess to use. Suggested crosslinker mol ar excesses are as follows (alsosee Table 1):? Proteinsamples < 1 mg/ml use 40-80-foldmolar excess.?Proteinsamplesof1-4 mg/ml use 20-foldmolarexcess.?Proteinsamplesof5-10 m

24、g/ml use 5- to10-fold mola r excess.Table 1. Crosslinker preparation and molar excess to use for1 ml of sample.Immediately before use, dissolvecrosslinker in the appropriate solvent at the concentration denote d inparentheses; then add the listed volume to a 1 ml protein sample. For example, to use

25、the No-Weigh Sulfo-SMCC, d issolvethe 2 mg contents of themicrotube in 200仙 lofbufferand then add the prescribedvolumeto per 1mlsample. For the other products, the appropriate amount of dry r eagent must be weighed on a balance ., mg Sulfo-SMCC for dissolution in 500 仙 l buffer).Protein-NH2 Concentr

26、ation(based on a 50 kDa protein) 10 mg/ml 1 mg/ml mg /mlCrosslinker Molar Excess 5X 20X 50X Sulfo-SMCC(in50mM sodiumphosphateorwater)100 川 mg/ml*)40 " l mg/ml*) 50 " l mg/ml*) No-Weigh Sulfo-SMCC(in50mM sodiumphosphateorwater)50 屋(10 mg/ml*) 20 膜 (10 mg/ml*) 25 膜 (10 m g/ml*) SMCC(in DMSO

27、or DMF)100lmg/ml*)100lmg/ml*)100lmg/ml*)*Concentration of each crosslinker before adding to protei n sample.Note: If the Sulfo-SMCC solution does not completely dissolve, place the tubeunder hot running wateror incubate for several minutes ina 50° C water bath.3. Incubate reaction mixturefor 30

28、 minutesat room temperature or 2 hours at 4° C.4. Remove excess crosslinkerusing a desalting column equilibrated with Conjugation Buffer.5. Combineand mix Protein-SH anddesalted Protein-NH2ina molar ratio corresponding tothatdesiredfor the finalconjugate and consistent with the relative number

29、of sulf hydryl and activated amines thatexist on thetwo proteins.6. Incubate thereaction mixture atroom temperaturefor 30 minutes or 2 hours at 4° C.Note: Generally, thereis noharmin allowing thereaction to proceed for several hoursor overnight,although usually the reaction will be complete in

30、the specified time. To stop the conjugation reaction before completion, add buffer containing reduced cysteine 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.Ad

31、ditional InformationA. Please visit the Pierce website for additional informa tion including the following item: ?Tech Tip: Attach an antibody onto glass, silica or quart z surface B. Two-step reaction schemeMaleimide-activated AntibodyAntibody-enzyme ConjugateSulfo-SMCCAntibodyAntibodyAntibodyEnzym

32、eEnzymeFigure 1. Two-stepreaction scheme forconjugating antibody and enzyme proteinswith Sulfo-SMCC. In this example, thecrosslinker is first reacted with the antibody to producea maleimide-activated protein. After excess non-reacted crossli nker and by-products are removed, the maleimide-activated

33、anti body is reacted with the appropriate molar ratio of enzyme having sulfhydryl groups. Usually, several or multiple maleimi de-activations occur per antibodymolecule,enablingseveralenzyme molecules to be conjugatedto each antibodymolecule.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NHSS旨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琉基交联。NHS舌性酯与伯胺在PH7-9的环境形成酰胺键。马来酰亚胺与琉基在的环境下形成稳定的硫醴 键。在水溶液中，NHS舌性酯的水解是（与氨基的反应）个竞争反应。马来酰亚 胺比NHS急定，1是在PH大于时，马来酰亚胺会慢慢水解，失去与琉基反应的 特异性。因而，在使用SMCC通常是在的环境下进行，并且先让NHS®生反应。 SMCCg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饰之后可以冻干存放一段时间