SMCC结构反应解释.pdf

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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;用 SM

2、CC 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 SMCC succinimidyl 4-N-maleimidomethylcyclohexane-1-carboxylate,50 mg Molecular 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-maleimidomethylcyclohexane-1-carboxylate,1 g Sulfo-SMCC,50 mg Sulfo-SMCC,No-Weigh Format,8 2 mg microtubes Molecular Weight:Spacer Arm:Net Mass Added:CAS:92921-24-9

4、 Storage:Upon receipt store desiccated at-20 C.Product is shipped at ambient temperature.Introduction SMCC and its water-soluble analog Sulfo-SMCC are heterobifunctional crosslinkers that contain N-hydroxysuccinimide NHS ester and maleimide groups that allow covalent conjugation of amine-and sulfhyd

5、ryl-containing molecules.NHS esters react with primary amines at pH 7-9 to form amide bonds,while maleimides react with sulfhydryl groups at pH to form stable thioether bonds.In aqueous solutions,NHS ester hydrolytic degradation is a competing reaction whose rate increases with pH.The maleimide grou

6、p is more stable than the NHS-ester group but will slowly hydrolyze and loses its reaction specificity for sulfhydryls at pH values .For these reasons,conjugations with these crosslinkers are usually performed at pH with the NHS-ester amine-targeted reacted before or simultaneous with the maleimide

7、sulfhydryl-targeted reaction.The cyclohexane ring in the spacer arm of these reagents decreases the rate of hydrolysis of the maleimide group compared to similar reagents that do not contain this This feature enables proteins that have been maleimide-activated with SMCC or Sulfo-SMCC to be lyophiliz

8、ed and stored for later conjugation to a sulfhydryl-containing molecule.Many maleimide-activated protein products are produced in this manner see Related Products.SMCC and Sulfo-SMCC are often used to prepare antibody-enzyme and hapten-carrier protein conjugates in a two-step reaction scheme.First,t

9、he amine-containing protein is reacted with 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 attached to the first protein.Sulfo-SMCC

10、 is soluble in water and many other aqueous buffers to approximately 10 mM,although solubility decreases with increasing salt concentration.SMCC is not directly water-soluble and must be dissolved in an organic solvent such as dimethylsulfoxide DMSO or dimethylformamide DMF;subsequent dilution into

11、aqueous reaction buffer is generally possible,and most protein reactants will remain soluble if the final concentration of organic solvent is less than 10%.SMCC and Sulfo-SMCC Important Product Information SMCC and Sulfo-SMCC are moisture-sensitive.Store reagent vial in desiccant.Equilibrate vial to

12、 room temperature before opening to avoid moisture condensation inside the container.Dissolve needed amount of reagent and use it immediately before hydrolysis occurs.Discard any unused reconstituted reagent.Do not store reagent in solution.No-Weigh Microtube Handling:Immediately before use,puncture

13、 the microtube foil with a pipette tip,add 200 l of 50 mM sodium phosphate buffer pH or ultrapure water and pipette up and down to mix.After use,cut the used microtube from the microtube strip and discard.Store the unused microtubes in the foil pouch provided.Note:Do not use phosphate-buffered salin

14、e PBS for initial 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.Avoid buffers containing primary amines.,Tris or glycine and sulfhydryls during conjugati

15、on,because they will compete with the intended reaction.If necessary,dialyze or desalt samples into an appropriate buffer such as phosphate-buffered saline PBS.Molecules to be reacted with the maleimide moiety must have free reduced sulfhydryls.Reduce peptide disulfide bonds with Immobilized TCEP Di

16、sulfide Reducing Gel Product No.77712.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,followed by two passes through a suitable desalting column.,Zeba Desalt Spin Columns.Be aware that proteins.,anti

17、bodies may be inactivated by complete reduction of their disulfide bonds.Selective reduction of hinge-region disulfide bonds in IgG can be accomplished with 2-MercaptoethylamineHCl 2-MEA,Product No.20408.Sulfhydryls can be added to molecules using N-succinimidyl S-acetylthioacetate SATA,Product No.2

18、6102 or 2-iminothiolaneHCl Trauts Reagent,Product No.26101,which modify primary amines.Procedure for Two-step Protein Crosslinking Generally,a 10-to 50-fold molar excess of crosslinker over the amount of amine-containing protein results in sufficient maleimide activation to enable several sulfhydryl

19、-containing proteins to be conjugated to each amine-containing protein.More dilute protein solutions require greater fold molar excess of reagent to achieve the same activation level.Empirical testing is necessary to determine optimal activation levels and final conjugation ratios for the intended a

20、pplication.A.Material Preparation Conjugation Buffer:phosphate-buffered saline PBS=100 mM sodium phosphate,150 mM sodium chloride,pH;.,Product No.28372 or other amine-and sulfhydryl-free buffer at pH see Important Product Information adding EDTA to 1-5 mM helps to chelate divalent metals,thereby red

21、ucing disulfide formation in the sulfhydryl-containing protein Desalting column to separate modified protein from excess crosslinker and reaction byproducts.,Zeba Desalt Spin Columns Amine-containing Protein-NH2 and sulfhydryl-containing proteins Protein-SH to be conjugated B.Protocol Note:For best

22、results,ensure that Protein-SH is prepared 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 protein solution.The concentration of the Protein-NH2 determines the crosslinker molar excess to use.Suggested cro

23、sslinker molar excesses are as follows also see Table 1:Protein samples 做 下一步；OD 值补加多肽,直至达到要求；OD 值返回多肽合成步骤重新质控;Ellman 试剂是用来检测游离巯基的,如果检测液显黄色说明多肽的 Cys 的巯基大部分以游离态存在；如果检测液不显黄色则说明多肽 Cys 中的巯基已经被氧化形成二聚体或多聚体;8、将多肽液滴加到 KLH-SMCC 管中,室温下用垂直混匀器混匀反应 4 小时;9、用 Ellman 试剂检测多肽中的巯基：在 96 孔板中加入 100 l Ellman 试剂储备液,再加入10 l 教练后的多肽溶液,用Nano分光光度计在=412 nm下测定紫外吸收值;OD 值则再补加 SMCC 活化好的 KLH 蛋白继续交联;如果 Ellman 试剂显黄色说明多肽与KLH 蛋白偶联不完全；如果 Ellman 试剂不显黄色则说明多肽已经全部与 KLH蛋白偶联