Sulfo-SMCC

合集下载

SOP Pep-KLH

多肽与KLH的连接－SH法1. 每瓶KLH蛋白(20mg，Pierce cat # 77600)中加入3ml纯水，轻轻摇晃使之溶解（不得剧烈振荡）。

2. 以纯水配制10 mg/ml的Sulfo-SMCC (Pierce cat # 22322)溶液。

3. 适量KLH蛋白溶液中按1：10（重量）加入Sulfo-SMCC溶液，轻轻摇晃混合后室温放置1小时。

4. 将G25 Sephadex凝胶过滤层析柱以连接缓冲液平衡（五倍柱体积）。

5. 将活化的KLH蛋白用G25 Sephadex凝胶过滤层析柱进行脱盐（上样体积控制在柱床的25％以内），以280nm紫外检测和收集流出液。

6.根据上样蛋白总量和收集溶液的体积估算活化的KLH浓度。

7.取少量干粉，以连接缓冲液进行可溶解性试验，如不容于连接缓冲液再用DMSO进行可溶解性试验（溶于DMSO则控制终浓度在25％以内并调节PH为7.2）。

如多肽不能溶解，则不进行以下实验，8.称取适量的多肽干粉：兔源多抗(2)：3mg小鼠源单抗(5): 2mg以连接缓冲液或DMSO（<30%）溶解使浓度为10mg/ml。

9. 将活化的KLH和溶解的多肽以1：1（重量）混合，在室温轻轻摇晃混合2小时。

10. 将进行了连接的混合物置透析袋对PBS进行透析过夜（用于免疫兔制备多抗可以免做）。

11. 将G25 Sephadex凝胶过滤层析柱以20％乙醇（五倍柱体积）平衡后保存。

12. 连接物进行SDS-PAGE以估算连接的效率。

附：连接缓冲液NaCl (0.9M) 52.6gNaH2PO4 (0.1M) 12.0g0.5M EDTA 200mls调节PH为7.2，以纯水补足至1L。

20％乙醇无水乙醇200ml纯水补足至1000mlPBS－NH2法1.取少量多肽干粉，以PBS缓冲液进行可溶解性试验，不可溶解不进行下面实验。

2.称取适量的多肽干粉：兔源多抗(2)：4mg小鼠源单抗(5): 2mg以PBS缓冲液溶解使浓度为5mg/ml。

交联剂的性质、作用、溶解性及其常见分类

交联剂的性质、作用、溶解性及其常见分类交联剂的性质、作用、溶解性及其常见分类交联剂是一种能在线型分子之间起架桥作用，从而使多个线型分子相互键合成网装结构的物质。

促进和调节聚合物分子链间共价键或离子键的物质。

常事分子间含有多个官能团的物质，如有机二元酸，多元醇等；或是分子内含有多个不饱和双键的化合物，如二乙烯基苯和二异氰酸酯,N,N-亚甲基双丙烯基酰胺等。

可以同单体一起投料，待缩聚到一定程度发生交联，使产物变为不溶的交联聚合物；也可在线型分子中保留一定的官能团，再加入特定的物质进行交联，如酚醛树脂的固化和橡胶的硫化。

交联剂的作用交联剂主要用在高分子材料中。

因为高分子材料的分子结构就像一条长长的线，没交联时强度低，易拉断，且没有弹性，交联剂的作用就是在线型分子间形成化学键，使线型分子相互连在一起，形成网状结构，这样提高橡胶的强度和弹性。

①多种热塑料（聚乙烯、聚氯乙烯、EVA，聚苯乙烯等）的交联和改性。

热交联一般的添加量为1~3%，另加过氧化二异丙苯0.2~1%，辐照交联添加量为0.5~2%。

交联后可显著提高产品的耐热性，阻燃性，耐熔性、及机械强度等。

②乙丙橡胶、各种氟橡胶、CPE等特种橡胶的助硫化，可显著的缩短硫化时间，提高强度、耐磨性、耐溶性和腐蚀性。

③丙烯酸、苯乙烯型离子交换树脂的交联。

他比二乙烯苯交联剂用量少、质量高、可制备抗污、强度大、大孔径、耐热、耐酸碱，抗氧化等性能极佳的离子型交换树脂。

④聚丙烯酸酯，聚烷基丙烯酸酯的改性。

可显著地提高耐热性、光学性能和工艺加工性能等。

典型用于普通有机玻璃的耐热改性。

⑤环氧树脂、DAP树脂的改性。

可提高耐热性、粘合性、机械强度和尺寸稳定性。

典型用于环氧灌封料和包封料的改性。

⑥不饱和聚酯和热塑性聚酯的交联和改性。

可显著的提高耐热性，抗化学腐蚀性，尺寸稳定性，耐候性和机械性能等。

典型用于提高热压性不饱和聚酯玻璃刚制品耐热性。

⑦TAI C本身的均聚物——聚三烯丙基异三聚氰酸酯是一种透明、硬质、耐热、电绝缘优良的树脂。

SMCC结构反应解释

SMCC就是一类含有N-羟基琥珀酰亚胺(NHS)活性酯与马来酰亚胺的双功能偶联剂、可以将分别含有巯基与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活性酯的水解就是(与氨基的反应)个竞争反应。

马来酰胺比NHS稳定,但就是在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时通常就是在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就是,SMCC怕潮湿,存放时要与干燥剂一起存放。

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-maleimidomethyl]cyclohexane-1-carboxylate), 50 m gMolecular Weight: 334、32 Spacer Arm: 8、3 Å Net Mass Added: 219、09Storage: Upon receipt store desiccated at 4° C、Product is shipped at ambient temperature、Sulfo-SMCC (sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxy late), 1 g Sulfo-SMCC, 50 mgSulfo-SMCC, No-Weigh™ Format, 8 × 2 mg microtubesMolecular Weight: 436、37 Spacer Arm: 8、3 ÅNet Mass Added: 219、09 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 heterobifunctional crosslin kers that contain N-hydroxysuccinimide (NHS) ester and maleimide groups that allow covalent conjugation of amine- and sulfhydryl-containing molecules、 NHS esters re act with primary amines at pH 7-9 to form amide bonds, while maleimides react with sulfhydryl groups at pH 6、5-7、5 to form stable thioether bonds、 In aqueous solutions, NHS ester hydrolytic degrad ation is a competing reaction whose rate increases with pH、 The maleimide group is more stable than the NHS-ester group but will slowly hydrolyze and loses its reaction specificity for sulfhydryls at pH values > 7、5、 For these reasons, conjugations with these crosslinkers are usually performed at pH 7、2-7、5, with the NHS-ester (amine-targeted) reacted before or simultaneous with the malei mide (sulfhydryl-targeted) reaction、The cyclohexane ring in the spacer arm of these reagents decreases the rate of hy drolysis of the maleimide group compared to similar reagents that do not contain this r ing、1 This feature enables proteins that have been maleimide-activated with SMCC or Sul fo-SMCC to be lyophilized 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, the amine-containing protein is reacted with a several-fold molar excess of the crosslinker, followed by rem oval of excess (nonreacted) reagent by desalting or dialysis; finally, the sulfhydryl-con taining molecule is added to react with the maleimide groups already attached to the fi rst protein、Sulfo-SMCC is soluble in water and many other aqueous buffers to approximatel y 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 dimeth ylsulfoxide (DMSO) or dimethylformamide (DMF); subsequent dilution into 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-SMCCImportant Product Information•SMCC and Sulfo-SMCC are moisture-sensitive、 Store reagent vial in desiccant 、 Equilibrate vial to room temperature before opening to avoid moisture condensatio n inside the container、 Dissolve needed amount of reagent and use it immediately be fore hydrolysis occurs、 Discard any unused reconstituted reagent、 Do not store rea gent in solution、•No-Weigh Microtube Handling: Immediately before use, puncture the microtube foil with a pipette tip, add 200 µl of 50 mM sodium phosphate buffer (pH 7、0-7、5) or ultrapure water and pipette up and down to mix、 After use, cut the used microt ube from the microtube strip and discard、 Store the unused microtubes in the foil po uch provided、Note: Do not use phosphate-buffered saline (PBS) for initial dissolution of Sulfo-SMCC; the reagent does not dissolve well in buffers exceeding 50 mM total salts、 H owever, once dissolved, the solution can be further diluted in PBS or other non-amine buffers、•Avoid buffers containing primary amines (e、g、, Tris or glycine) and sulfhydryls during conjugation, because they will compete w ith 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) sulf hydryls、 Reduce peptide disulfide bonds with Immobilized TCEP Disulfide Reducin g Gel (Product No、 77712)、 For proteins, reduce disulfide bonds using 5 mM TCE P (1:100 dilution of Bond-Breaker® TCEP Solution, Product No、 77720) for 30 min utes at room temperature,followed by two passes through a suitable desalting column (e、g、, Zeba™ Desalt Spin Columns)、 Be aware that proteins (e、g、, antibodies) may be inactivated by complete reduction of their disulfide bonds、Selective reduction of hinge-region disulfide bonds in IgG can be accomplished with 2-Mercaptoethylamine•HCl (2-MEA, Product No、 20408)、 Sulfhydryls can be add ed to molecules using N-succinimidyl S-acetylthioacetate (SATA, Product No、 2610 2) or 2-iminothiolane•HCl(Traut’s Reagent, Product No、 26101), which modify pri mary amines、Procedure for Two-step Protein CrosslinkingGenerally, a 10- to 50-fold molar excess of crosslinker over the amount of amine -containing protein results in sufficient maleimide activation to enable several sulfhyd ryl-containing proteins to be conjugated to each amine-containing protein、 More dil ute protein solutions require greater fold molar excess of reagent to achieve the same activation level、 Empirical testing is necessary to determine optimal activation level s and final conjugation ratios for the intended application、 A、 Material Preparation •Conjugation Buffer: phosphate-buffered saline (PBS = 100 mM sodium phosphat e, 150 mM sodium chloride, pH 7、2; e、g、, Product No、 28372) or other amine- and sulfhydryl-free buffer at pH 6、5-7、5 (see Important Product Information) – adding EDTA to 1-5 mM helps to chelate div alent metals, thereby reducing disulfide formation in the sulfhydryl-containing protein • Desalting column to separate modified protein from excess crosslinker and reac tion byproducts (e、g、, Zeba Desalt Spin Columns)•Amine-containing (Protein-NH2) and sulfhydryl-containing proteins (Protein-SH ) to be conjugatedB、 ProtocolNote: For best 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 con centration of the Protein-NH2 determines thecrosslinker molar excess to use、 Suggested crosslinker molar excesses are as fo llows (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 molar excess、Table 1、 Crosslinker preparation and molar excess to use for 1 ml of sample、 I mmediately before use, dissolve crosslinker in the appropriate solvent at the concentra tion denoted in parentheses; then add the listed volume to a 1 ml protein sample、 For example, to use the No-Weigh Sulfo-SMCC, dissolve the 2 mg contents of the microt ube in 200 µl of buffer and then add the prescribed volume to per 1 ml sample、 For t he other products, the appropriate amount of dry reagent must be weighed on a balanc e (e、g、, 2、4 mg Sulfo-SMCC for dissolution in 500 µl buffer)、Protein-NH2 Concentration(based on a 50 kDa protein) 10 mg/ml 1 mg/ml 0、5 mg/mlCrosslinker Molar Excess 5X 20X 50X Sulfo-SMCC(in 50 mM sodium phosphate or water) 100 µl (4、8 mg/ml*) 40 µl (4、8 mg/ml*) 50 µl (4、8 mg/ml*) No-Weigh Sulfo-SMCC(in 50 mM sodium phosphate or water)50 µl (10 mg/ml*) 20 µl (10 mg/ml*) 25 µl (10 mg/ml*) SMCC(in DMSO or DMF)100 µl (3、7 mg/ml*)100 µl (1、5 mg/ml*)100 µl (1、8 mg/ml*)*Concentration of each crosslinker before adding to protein sample、Note: If the Sulfo-SMCC solution does not completely dissolve, place the tube under hot running water or incubate for several minutes in a 50°C water bath、3、 Incubate reaction mixture for 30 minutes at room temperature or 2 hours at 4°C、4、 Remove excess crosslinker using a desalting column equilibrated with Conju gation Buffer、5、 Combine and mix Protein-SH and desalted Protein-NH2 in a molar ratio corr esponding to that desired for the finalconjugate and consistent with the relative number of sulfhydryl and activated am ines that exist on the two proteins、 6、 Incubate the reaction mixture at room temper ature for 30 minutes or 2 hours at 4°C、Note: Generally, there is no harm in allowing the reaction to proceed for several hours or overnight, although usually the reaction will be complete in the specified tim e、 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: Conjugation efficiency can be estimated by electrophoresis separation and subs equent protein staining、Additional InformationA、 Please visit the Pierce website for additional information including the follo wing item: •Tech Tip: Attach an antibody onto glass, silica or quartz surface B、 Two-step re action schemeMaleimide-activated AntibodyAntibody-enzyme ConjugateSulfo-SMCCAntibodyAntibodyAntibodyEnzymeEnzymeFigure 1、 Two-step reaction scheme for conjugating antibody and enzyme prote ins with Sulfo-SMCC、 In this example, the crosslinker is first reacted with the antib ody to produce a maleimide-activated protein、 After excess non-reacted crosslinker and by-products are removed, the maleimide-activated antibody is reacted with the ap propriate molar ratio of enzyme having sulfhydryl groups、 Usually, several or multip le maleimide-activations occur per antibody molecule, enabling several enzyme molec ules to be conjugated to each antibody 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。

氨基磺酸氨红外光谱_理论说明

氨基磺酸氨红外光谱理论说明1. 引言1.1 概述氨基磺酸氨是一种重要的有机化合物，具有广泛的应用领域。

它在医药、农业、食品工业等方面均有重要作用。

红外光谱是研究化学物质结构和分子振动特性的常用手段之一。

本文旨在通过理论说明，深入探讨氨基磺酸氨红外光谱的产生机制及其与分子结构的关系。

1.2 文章结构本文包括以下几个部分：引言、氨基磺酸氨红外光谱理论说明、实验方法与仪器设备、实验结果与分析以及结论与展望。

首先介绍了文章的背景和概述，然后详细阐述了氨基磺酸氨红外光谱的基本原理，探讨了其结构特点以及产生机制。

接下来介绍了实验所采用的方法和使用的仪器设备，并对实验结果进行了解析和分析。

最后总结实验结果，并展望其在未来的应用前景。

1.3 目的本文旨在通过理论说明，对氨基磺酸氨红外光谱进行深入研究，探索其产生机制以及与分子结构的关系。

通过实验方法和仪器设备的介绍，结合实验结果与分析，进一步理解氨基磺酸氨红外光谱的特性和应用前景。

此外，本文也致力于为相关领域的研究提供理论指导和实验依据。

以上就是对文章“1. 引言”部分内容的详细清晰撰写，请核对确认是否符合要求。

2. 氨基磺酸氨红外光谱理论说明：2.1 红外光谱的基本原理：红外光谱是一种常用的分析技术，它通过测量物质在红外辐射下的吸收和散射来获取样品的结构信息。

红外辐射穿过样品后，被吸收能量与样品中化学键振动相关联。

这些振动会引起红外光谱图上的特定频率出现吸收峰，进而提供了样品组成和结构信息。

2.2 氨基磺酸的结构特点：氨基磺酸是一种含有氨基和磺酸基团的有机化合物，其分子式为（NH2）2SO2。

它在化学结构上包含着两个氨基（NH2）和一个磺酸基团（SO2），由于这些功能团在分子中的不同部位以及相互之间的取向而呈现出不同的振动模式。

2.3 氨基磺酸氨红外光谱的产生机制：当氨基磺酸置于红外辐射下时，它会与辐射作用并发生吸收。

氨基磺酸分子中的化学键振动以及分子内部不同部位的转动和振动等引发物质对红外光谱的吸收。

基因工程纳米颗粒在EB病毒疫苗研发中的应用概述

基因工程纳米颗粒在EB病毒疫苗研发中的应用概述1 引言EB 病毒(Epstein-Barr Virus，EBV)是人类至今未攻克的一大科学难题。

该病毒在人群中感染非常广泛，流行病学研究表明全球约有90% 的成年人存在EB 病毒感染。

大多数初次感染发生在幼儿时期且无临床症状，而青少年时期的感染往往会引发传染性单核细胞增多症。

此外，成年人感染EB 病毒可诱发多种疾病甚至癌症，全球每年新增癌症病例中就有约20 万病例与EB 病毒感染直接相关，因此EB 病毒被世界卫生组织归为第一类致癌病毒。

与EB 病毒感染高度相关的癌症包括鼻咽癌、胃癌、霍奇金(Hodgkin’s)淋巴瘤、伯基特(Burkitt’s)淋巴瘤等。

其中，鼻咽癌与EB 病毒相关性最高，并且高发于我国广东、香港等南方地区，被列为我国重点防治的十大恶性肿瘤之一。

目前，全球还没有可接种的EB 病毒防治疫苗。

而在我国，针对高发的EB 病毒阳性鼻咽癌，主要手段为放射性疗法同多种化疗药联合治疗，而针对晚期鼻咽癌患者的治疗手段则更为有限。

因此，开发防治EB 病毒感染所诱发相关疾病的变革性疫苗刻不容缓。

随着合成生物学、基因工程技术的进步，各种工程化合成疫苗载体得以迅速发展，并在EB 病毒预防及相关肿瘤综合治疗中发挥着越来越重要的作用。

2 EB 病毒EB 病毒主要通过唾液在鼻咽部及扁桃体传播感染，在鼻咽上皮细胞以及随上皮组织中的血管到达B 淋巴细胞中增殖复制或进入潜伏期。

EB 病毒基因组主要以附加体形式(Episomes)游离于宿主细胞的细胞核并编码约100 个基因[3]。

EB 病毒存在多种变异株，因此引发的相关疾病具有非常明显的地域差异。

例如，伯基特淋巴瘤高发于中非地区，霍奇金淋巴瘤高发于欧美国家，鼻咽癌高发于我国南方地区。

通过大量比对华南高发区与欧美地区及其他低发区鼻咽癌样本中EB 病毒的全基因组捕获测序，分析鼻咽癌发生与相应EB 病毒基因组的关系，证实广东地区约83% 的鼻咽癌患者感染的EB 病毒高危亚型为BALF2基因非同义变异位点BALF2_CCT，而并非普遍感染中国人群的LMP1-China1 亚株[4]，该亚型在欧洲和非洲等地则极为罕见。

一种巯基化ssDNA探针-功能化修饰的MOFs复合物材料及其制备方法和应

(19)中华人民共和国国家知识产权局(12)发明专利申请(10)申请公布号 (43)申请公布日 (21)申请号 202011344020.2(22)申请日 2020.11.25(71)申请人西安交通大学地址 710049 陕西省西安市咸宁西路28号(72)发明人吴春生　陈雅婷　田玉兰　朱平　刘淑阁　杜立萍　陈炜　(74)专利代理机构西安通大专利代理有限责任公司 61200代理人安彦彦(51)Int.Cl.G01N 21/64(2006.01)(54)发明名称一种巯基化ssDNA探针-功能化修饰的MOFs复合物材料及其制备方法和应用(57)摘要本发明公开了一种巯基化ssDNA探针‑功能化修饰的MOFs复合物材料及其制备方法和应用，该复合物材料包括MOFs材料UiO ‑66‑NH2，双功能交联试剂Sulfo ‑SMCC，巯基化ssDNA探针以及荧光染料RhoB等部分。

本方法首先实现巯基化ssDNA探针‑功能化修饰的MOFs复合物材料的制作；该复合材料的TEM图和Zeta电位表征；该复合物材料对液态醇类的特异性检测；以乙醇为代表的检测限检测以及可重复性实验。

结果表明，巯基化ssDNA探针‑功能化修饰的MOFs复合物材料实现了对液态醇类的简单有效，成本低廉，高灵敏度检测，为液态醇类的便携式检测设备的发展提供了新的研究方向。

权利要求书2页说明书6页附图3页CN 112683862 A 2021.04.20C N 112683862A1.一种巯基化ssDNA探针‑功能化修饰的MOFs复合物材料，其特征在于，包括MOFs材料UiO‑66‑NH2，双功能交联试剂Sulfo‑SMCC，巯基化ssDNA探针以及荧光染料RhoB；所述巯基化ssDNA探针通过双功能交联试剂Sulfo‑SMCC共价修饰到MOFs材料UiO‑66‑NH2表面，形成巯基化ssDNA探针‑功能化修饰的MOFs材料，然后利用MOFs材料UiO‑66‑NH2的高孔径率和吸附性，将荧光染料RhoB装载进巯基化ssDNA探针‑功能化修饰的MOFs材料的孔洞中，形成巯基化ssDNA探针‑功能化修饰的MOFs复合物材料。

一种在氨基磁珠表面共价偶联蛋白质的方法[发明专利]

[19]中华人民共和国国家知识产权局[12]发明专利申请公布说明书[11]公开号CN 101348517A[43]公开日2009年1月21日[21]申请号200810115278.8[22]申请日2008.06.20[21]申请号200810115278.8[71]申请人北京倍爱康生物技术有限公司地址100070北京市丰台区海鹰路1号院6号楼[72]发明人仝文斌陈立杰雷焱 [51]Int.CI.C07K 1/10 (2006.01)权利要求书 1 页说明书 4 页附图 2 页[54]发明名称一种在氨基磁珠表面共价偶联蛋白质的方法[57]摘要一种在氨基磁珠表面共价偶联蛋白质分子的方法，该法用不同的活化剂分别对氨基磁珠和蛋白质分子进行活化，之后将二者混合即可将蛋白质分子高效率共价偶联在磁珠表面。

由于活化后蛋白质分子和磁珠具有不同的活化集团，彼此之间不会发生反应，从而有效的避免了蛋白质分子之间或磁珠之间的聚集现象。

此外由于活化产生的巯基和马来酰亚胺活化集团之间的反应迅速高效，保证了该方法有很高的偶联效率和良好重复性。

偶联后磁珠可广泛用于免疫检测，细胞分选等领域。

200810115278.8权利要求书第1/1页 1.一种在氨基磁珠表面共价偶联蛋白质的方法，其特征在于，实现步骤包括：(1)氨基磁珠的活化：混悬在磁珠活化缓冲液内磁珠表面的氨基(NH2-)与活化剂发生共价反应生成含马来酰亚胺的化学集团。

(2)蛋白质的活化：溶解在蛋白质活化缓冲液内的蛋白质的氨基与活化剂发生共价反应生成巯基(SH-)。

(3)偶联：活化磁珠和蛋白质在磁珠偶联缓冲液内混合后通过马来酰亚胺与巯基的共价反应蛋白质被共价偶联在磁珠表面。

(4)稀释和保存：将偶联后磁珠用磁珠保存缓冲液稀释，4℃保存。

(4)偶联效果检测：利用酶联免疫测定法原理检测偶联效果。

2.根据权利要求1所述的一种在氨基磁珠表面共价偶联蛋白质的方法，其特征在于，所述的氨基磁珠活化剂为Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate(SMCC)或Sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate(Sulfo-SMCC)。

16多肽与载体偶联——三种不同介导方式

一、Frdbio –SH介导多肽与载体偶联1. cKLH载体蛋白与sulfo-SMCC的偶联（以偶联20mg多肽为例，根据实验实际偶联量，所有试剂体积作同比缩放）1.1 称取20mg cKLH（Frdbio，Cat No.: BCJ0002），溶于2ml超纯水，配成【10 mg/ml cKLH溶液】。

1.2 称取10 mg Sulfo-SMCC(Frdbio)于2ml超纯水配成【5mg/ml Sulfo-SMCC溶液】。

1.3 将以上两种溶液等体积混匀，室温(25℃)反应60 min或者37℃反应30 min，磁力搅拌器慢速均匀搅动反应，避免产生气泡。

1.4 反应完后将以上反应溶液装入10kD透析袋，在PBS（pH7.2）溶液中透析除去过多Sulfo-SMCC，每隔3h换液，至少换3～4次，确保透析完全，即为【活化的cKLH载体】（有条件的也可以用sephdex-G25分子筛色谱分离或超滤管。

活化的cKLH要尽快使用，不可久放。

）2. 多肽的偶联偶联前需检测多肽中-SH活性。

2.1 Ellman试剂法检测多肽-SH状态:方法：在96孔酶标板中，10μl多肽+100μl Ellman试剂，用分光光度计在96孔酶标板中412nm进行测量，OD ＞0.15时，多肽SH正常，如果OD＜0.15，说明多肽被氧化或者自我交联，不可使用。

偶联完成之后，透析前用上述同样方法检测DO＜0.03时，说明多肽已经80%以上全部偶联，可继续添加多肽；如果OD＞0.03，说明多肽过量未全部偶联。

如果没有Nano分光光度计，直接观察颜色，颜色变黄，说明游离—SH过量。

2.2 称取20mg多肽溶解于5ml交联缓冲液(0.1M PB，0.15M NaCl)中，配成【4mg/ml的多肽溶液】（对于难溶肽，可用≤30%的DMSO溶解），一般我们只偶联5～10mg多肽，等比例缩小体积即可。

2.3 将第1.4步透析好的cKLH与第5步配好的【4mg/ml多肽溶液】混合，室温(25℃)4h。

硅纳米粒子的功能化及生物分析应用

第60卷第3期2021年5月Vol.60No.3May 2021中山大学学报（自然科学版）ACTASCIENTIARUM NATURALIUM UNIVERSITATISSUNYATSENI硅纳米粒子的功能化及生物分析应用*李春荣1，3，邹小勇1，戴宗21.中山大学化学学院，广东广州5102752.中山大学生物医学工程学院，广东深圳5181073.黔南民族医学高等专科学校，贵州都匀558013摘要：硅纳米粒子作为一类新兴的荧光纳米材料在生物传感研究方面有许多优势。

近年来，开展功能化硅纳米粒子修饰在生物传感器、生化分析、荧光探针等方面受到科研工作者的广泛关注。

本综述对硅纳米粒子的功能化修饰技术，及其在荧光检测、生物传感、成像分析等领域的研究进展进行了总结和评述，并对硅纳米粒子的功能化发展前景及应用进行了展望。

关键词：硅纳米粒子；细胞成像；功能化修饰；生物传感；荧光检测中图分类号：O657文献标志码：A文章编号：0529-6579（2021）03-0001-11Founctional silicon nanoparticles and bioanalitical applicationLI Chunrong 1，3，ZOU Xiaoyong 1，DAI Zong 21.School of Chemistry ，Sun Yat -sen University ，Guangzhou 510275，China2.School of Biomedical Engineering ，Sun Yat -sen University ，Shenzhen 518107，China3.Qiannan Medical College for Nationalities ，Duyun 558013，ChinaAbstract ：As a newly emerging nanomaterial ，silicon nanoparticle possesses many advantages in the ap⁃plication of biosensor.In recent years ，silicon nanoparticles have been received widespread attention in biosensor ，bioanalytical ，and fluorescence probe.Herein ，the functional modification ，and application in fluorescence detection ，biosensor ，and imaging analytical of silicon nanoparticles were reviewed.Moreover ，the future functional modification developments and application of silicon nanoparticles are al⁃so discussed.Key words ：silicon nanoparticles ；cell imaging ；function modification ；biosensor ；fluorescence detection 硅是地壳中含量第二大元素，为各种硅相关应用材料提供了丰富而低成本的资源支持。

盐酸克伦特罗多克隆抗体和量子点的偶联

盐酸克伦特罗多克隆抗体和量子点的偶联金艳丹;栗慧;张岩蔚;魏东【摘要】为在制备盐酸克伦特罗和水溶性羧基量子点偶联物,给荧光快速检测技术的研究提供良好的依据.实验采用EDC/NHS两种偶联剂将二者共价偶联到一起,并经紫外扫描法、荧光扫描、斑点法、荧光免疫分析法对二者偶联效果进行了鉴定,结果表明,量子点和抗体偶联成功,偶联后吸收峰变宽、吸光度变大、荧光强度增加、抗体效价稍低,但不影响试验结果,可为盐酸克伦特罗药物的多残留试纸条及试剂盒的开发提供良好的参考依据.【期刊名称】《中国兽医杂志》【年(卷),期】2018(054)001【总页数】4页(P43-46)【关键词】量子点;盐酸克伦特罗多克隆抗体;偶联物【作者】金艳丹;栗慧;张岩蔚;魏东【作者单位】河北省农产品安全检测重点实验室,河北张家口075000;河北北方学院食品安全研究中心,河北张家口075000;河北省农产品安全检测重点实验室,河北张家口075000;河北北方学院食品安全研究中心,河北张家口075000;河北省农产品安全检测重点实验室,河北张家口075000;河北北方学院食品安全研究中心,河北张家口075000;河北省农产品安全检测重点实验室,河北张家口075000;河北北方学院食品安全研究中心,河北张家口075000【正文语种】中文【中图分类】Q657.39+盐酸克伦特罗又称“瘦肉精”(CL)，是白色或类白色晶体粉状，无嗅、味微苦，化学性质较为稳定，属于苯乙醇胺类β2肾上腺类神经兴奋剂。

它有较强的药性，化学性质稳定，溶解代谢率低，不易排出，残留量较高，大量用在饲料中可增强脂肪分解，肉迅速增长，大大提高了瘦肉率，增加瘦肉产量，虽说农药、饲料添加剂、动物激素等在一定剂量范围内使用，可以为畜牧业和经济的发展起一定的促进作用，但当其在生物体内含量超过临床用量的5～10倍时，便会导致畜禽中毒和大量药物残留，带来了相应的安全隐患[1]。

研究表明，过多食用含有瘦肉精的肉制品对人体某些机能损害极大，易使人出现肌肉震颤、心悸、呕吐等症状，特别是对高血压、心脏病、甲亢和前列腺肥大等疾病患者危害更大，严重的可导致死亡[2]。

医用耦合剂的成分

医用耦合剂的成分医用耦合剂是一种广泛应用于医疗领域的化学物质，其主要作用是将不相容的成分结合在一起，以便在医学测试和研究中更好地使用。

在医用耦合剂的制备过程中，其成分是一个非常重要的因素，它不仅影响着产品的质量和效果，还直接关系到其安全性和可靠性。

因此，本文将就医用耦合剂的成分进行详细介绍，以期更好地了解该化学物质。

一、医用耦合剂的定义医用耦合剂是一种可在体外将两种或两种以上的化学物质结合在一起的化学物质。

它主要由两种成分组成，即活性基团和耦合剂。

其中，活性基团是指具有特定化学性质的分子结构，它能够与其他分子结构发生化学反应，从而使它们结合在一起。

而耦合剂则是一种具有两个或两个以上活性基团的分子，它能够将不同的分子结合在一起，从而形成一个新的化合物。

二、医用耦合剂的特点医用耦合剂具有以下几个特点：1、高效性：医用耦合剂能够在较短的时间内将不同的化学物质结合在一起，从而提高研究和测试的效率。

2、稳定性：医用耦合剂能够在体外稳定存在，并且不会被其他化学物质影响。

3、可靠性：医用耦合剂的结合效果可靠，能够确保测试和研究结果的准确性和可靠性。

4、安全性：医用耦合剂不会对人体产生负面影响，具有较高的安全性。

三、医用耦合剂的主要成分医用耦合剂的主要成分包括活性基团和耦合剂，具体如下：1、活性基团活性基团是医用耦合剂的重要组成部分，其种类繁多，常见的有以下几种：（1）氨基基团：氨基基团是最常用的活性基团之一，能够与羧基、酰胺基等结构发生反应，形成多肽、蛋白质等化合物。

（2）羧基基团：羧基基团是一种含有羧酸基的分子，能够与氨基、醇基等结构发生反应，形成酯、酰胺等化合物。

（3）硫醇基团：硫醇基团是一种含有硫的有机化合物，能够与另一个含有硫醇基团的分子结合在一起，形成二硫键。

（4）双酰亚胺基团：双酰亚胺基团是一种含有酰亚胺结构的分子，能够与氨基、醇基等结构发生反应，形成多肽、蛋白质等化合物。

2、耦合剂耦合剂是医用耦合剂中的另一个重要成分，其种类较多，常见的有以下几种：（1）EDC：EDC是一种含有1-（3-二甲基氨基丙基）-3-乙基碳二亚胺结构的分子，能够将羧基和氨基结合在一起，形成酰胺键。

SMCC-结构反应解释

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。

INSTRUCTIONSSMCC (succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxy late), 50 mgMolecular Weight: Spacer Arm: ÅNet Mass Added:Storage: Upon receipt store desiccated at 4° C.Product is shipped at ambient temperature.Sulfo-SMCC (sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexan e-1-carboxylate), 1 g Sulfo-SMCC, 50 mgSulfo-SMCC, No-Weigh™Format, 8 × 2 mg microtubesMolecular Weight: Spacer Arm: ÅNet Mass 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 conjugation of amine- and sulfhydryl-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 st able thioether bonds. In aqueous solutions, NHS ester hydroly tic degradation is a competing reaction whose rate increases with pH. The maleimide group is more stable than the NHS-ester group but will slowly hydrolyze and loses its reaction specificity for sulfhydryls at pH values > . For these re asons, conjugations with these crosslinkers are usually performed at pH with the NHS-ester (amine-targeted) reacted befor e or simultaneous with 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 Sulfo-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 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 already attached 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 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-SMCCImportant Product Information•SMCC and Sulfo-SMCC are moisture-sensitive. Store reagent vial in desiccant. Equilibrate vial to room temperature befor e opening to avoid moisture condensation inside the container . Dissolve needed amount of reagent and use it immediately before hydrolysis occurs. Discard any unused reconstituted rea gent. Do not store reagent in solution. •No-Weigh Microtube Handling: Immediately before use, punctu re the microtube foil with a pipette tip, add 200 µl of 50 mM sodium phosphate buffer (pH or ultrapure water and pipette up and down to mix. After use, cut the used microt ube from the microtube strip and discard. Store the unused microtubes in the foil pouch provided.Note: 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. •Avoid buffers containing primary amines ., Tris or glycin e) and sulfhydryls during conjugation, because they will comp ete with the intended reaction. If necessary, dialyze or des alt 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. Reduce 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,followed by two passes through a suitable desalting colum n ., Zeba™Desalt Spin Columns). Be aware that proteins ., antibodies) may be inactivated by complete reduction of the ir disulfide bonds. Selective reduction of hinge-region disulf ide bonds in IgG can be accomplished with 2-Mercaptoethylamin e•HCl (2-MEA, Product No. 20408). Sulfhydryls can be added to molecules using N-succinimidyl S-acetylthioacetate (SATA, Pr oduct No. 26102) or 2-iminothiolane•HCl (Traut’s Reagent, Pr oduct No. 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-containing protein results in suffic ient maleimide activation to enable several sulfhydryl-containi ng 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 determine optimal activation lev els and final conjugation ratios for the intended application . A. Material Preparation •Conjugation 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 pH (see Important Product Information) –adding EDTA to 1-5 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 (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. 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 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 to use for 1 ml of sample. Immediately before use, dissolve cross linker in the appropriate solvent at the concentration denote d in parentheses; then add the listed volume to a 1 ml pr otein sample. For example, to use the No-Weigh Sulfo-SMCC, d issolve the 2 mg contents of the microtube in 200 µl of buffer and then add the prescribed volume to per 1 ml samp le. For the other products, the appropriate amount of dry r eagent must be weighed on a balance ., mg Sulfo-SMCC for dissolution in 500 µl 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 sodium phosphate or water) 100 µl mg/ml*) 40 µl mg/ml*) 50 µl mg/ml*) No-Weigh Sulfo-SMCC(in 50 mM sodium phosphate or water)50 µl (10 mg/ml*) 20 µl (10 mg/ml*) 25 µl (10 m g/ml*) SMCC(in DMSO or DMF)100 µl mg/ml*)100 µl mg/ml*)100 µl mg/ml*)*Concentration of each crosslinker before adding to protei n sample.Note: If the Sulfo-SMCC solution does not completely diss olve, place the tube under hot running water or incubate fo r several minutes in a 50°C water bath.3. Incubate reaction mixture for 30 minutes at room temp erature or 2 hours at 4°C.4. Remove excess crosslinker using a desalting column equ ilibrated with Conjugation Buffer.5. Combine and mix Protein-SH and desalted Protein-NH2 ina 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 temperature 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 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.Additional 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-SMCCAntibodyAntibodyAntibodyEnzymeEnzymeFigure 1. Two-step reaction scheme for conjugating antibod y and enzyme proteins with Sulfo-SMCC. In this example, the crosslinker 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 anti body is reacted with the appropriate molar ratio of enzyme having sulfhydryl groups. Usually, several or multiple maleimi de-activations occur per antibody molecule, enabling several e nzyme molecules to be conjugated to each antibody 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。

多肽和KLH偶联(1)

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。

马来酰亚胺比NHS稳定，但是在PH大于7.5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应，反应结束后通过脱盐柱或者透析的方法除掉没有反应完的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。

1、将20 mg SMCC（这是联接40条多肽的量）溶于2 ml DMF。

2、将0.8 ml KLH加入到25 ml圆底烧瓶中，补加1×PBS(pH 7.2)使蛋白终浓度为15mg/ml。

3、将溶解好的SMCC溶液缓慢滴加到120 mg KLH 蛋白体系中，室温搅拌反应1h。

4、用1 L 1×PBS (PH 7.4) 溶液于4℃下透析6 小时，除去游离的SMCC。

5、将透析后的KLH蛋白倒入50 ml离心管中，通过离心管的刻度确定其体积，根据反应前加入的KLH蛋白的量来计算透析后蛋白的浓度，然后根据其浓度将2.5 mg KLH-SMCC溶液转移到5 ml离心管中。

常用交联剂——精选推荐

常⽤交联剂什么是交联剂交联剂是⼀类⼩分⼦化合物，分⼦量⼀般在200-600之间，具有2个或者更多的针对特殊基团（氨基、巯基等）的反应性末端，可以和2个或者更多的分⼦分别偶联从⽽使这些分⼦结合在⼀起。

在⽣命科学研究中，巧妙地运⽤交联剂可以使很多⼯作取得突破。

交联剂的应⽤交联剂已经被⼴泛地应⽤于：细胞膜结构研究，蛋⽩质结构研究，蛋⽩质间相互作⽤研究，⽣物导弹研究，载体蛋⽩与半抗原的连接，蛋⽩质或其他分⼦的固相化，抗体的标记，标记转移，蛋⽩质与核酸的连接。

如何选择交联剂选择交联剂时要综合考虑这些因素：反应指向，间臂长度，⽔溶性，透膜性，可否切断，可否碘化。

PIERCE交联剂的特点· 种类最多，成熟的产品多达86种，可以满⾜各种偶联需要。

· ⽣理条件下即可共价交联，交联反应快速简便。

· 多种反应性基团可供选择，反应指向既可以有专⼀性也可以⽆专⼀性。

· 含有不同长度的间臂，有效地降低空间位阻效应。

· 部分产品内置可裂解基团，⼤⼤增强了交联剂应⽤的灵活性。

PIERCE交联剂⼀览以下是PIERCE交联剂⼀览表，⾄于每⼀种交联剂的全称、分⼦式、分⼦量、间臂长度、特征优点、包装规格等详细资料，请参阅PIERCE英⽂版⽬录或登录查阅。

在此特别推荐Greg T.Hermanson先⽣的专著，全书728页，⽬录号#20002。

221510ABH22101AEDP巯醇22295AMAS21451ANB-NOS27720APDP*巯醇20108APG21512ASBA*21564BASED*巯醇22331BMB22332BMDB⾼碘酸22330BMH22323BMOE22296BMPA22297BMPH22298BMPS22336BM[PEO]322337BM[PEO]421600BSOCOES碱21580BS320320DCC21525DFDNB20663DMA21666DMP20700DMS21702DPDPB巯醇20593DSG22585DSP巯醇21555DSS20589DST⾼碘酸20665DTBP巯醇22335DTME巯醇21578DTSSP巯醇22980EDC21565EGS羟胺22306EMCA22106EMCH22308EMCS22309GMBS22334HBVS22211KMUA22111KMUH22362LC-SMCC21651LC-SPDP巯醇22311MBS22305MPBH22605MSA27714NHS-ASA*22301PDPH巯醇28100PMPI21533SADP巯醇33030SAED巯醇21549SAND巯醇22600SANPAH27716SASD*巯醇26102SATA26100SATP22339SBAP27719SFAD巯醇22349SIA22329SIAB22360SMCC22416SMPB22363SMPH21558SMPT巯醇23013SPB21857SPDP巯醇20591Sulfo-DST⾼碘酸21566Sulfo-EGS羟胺22307Sulfo-EMCS22324Sulfo-GMBS21563Sulfo-HSAB21111Sulfo-KMUS21568Sulfo-LC-SMPT巯醇31650Sulfo-LC-SPDP巯醇22312Sulfo-MBS27735Sulfo-NHS-LC-ASA*21553Sulfo-SADP巯醇22589Sulfo-SANPAH22327Sulfo-SIAB22322Sulfo-SMCC22317Sulfo-SMPB33033Sulfo-SBED**巯醇22299TFCS22607THPP33043TMEA***33063TSAT**** 可以被碘化。

Sulfo-SMCC

Coupling of Peptide to KLHA. －SH1. Reconstitute one vial of carrier protein by adding 2 ml of ultrapure water to make a 10 mg/ml solution.Note: mcKLH forms a suspension that typically appears translucent to whitish blue. Do not vortex or heat the suspension, which will cause the mcKLH to precipitate.2.Immediately before use prepare a ~10 mM solution of Sulfo-SMCC in ultrapure water (5mg/ml).3.Add the appropriate volume of Sulfo-SMCC to the carrier protein as follows: 2 ml for mcKLH;3 ml for BSA;4 ml forOV A.4.Incubate for 60 minutes at room temperature or 30 minutes at 37°C with periodic gentle mixing.e a desalting column equilibrated with Conjugation Buffer to remove excess cross-linker.Note: The protocol is designed to yield effective immunogens for a wide variety of haptens but is not necessarily optimal for a specific hapten. Differences in size and structure of haptens will affect conjugation efficiencies. Using a molar excess of hapten over the carrier protein ensures efficient conjugation. Generally, reacting equal mass amounts of hapten and carrier protein will achieve sufficient molar excess. If a molar excess of hapten is not available, add asulfhydryl-containing compound, such as cysteine, after conjugation to quench any remaining active maleimide groups.6.Dissolve up to 20 mg of the sulfhydryl-containing hapten in 5 ml of Imject Maleimide Conjugation Buffer.Note: For haptens with limited solubility, DMSO may be used for solubilization. Use ≤30% DMSO in the final conjugation solution or the carrier protein may irreversibly denature.7.Immediately mix the peptide and activated carrier protein and react for 2 hours at room temperature.8.Purify conjugate by desalting or dialysis to remove EDTA and sodium azide.Additional Notes:* PBS may be used for conjugate purification. If the conjugate will be frozen, use the Purification Buffer Salts, which willpreserve the product during freeze-thaw cycles.* Desalting or dialysis will not separate non-conjugated protein; however, a large excess of hapten is used in this protocol,making it unlikely that non-conjugated carrier exists in significant quantity.* If DMSO was used in the conjugation, add DMSO to the Purification Buffer Salts for desalting to prevent precipitation in the column; dialysis is not compatible with DMSO.* If a precipitate has formed during conjugation, centrifuge the material, collect the supernatant and save the e only the supernatant for purification. Combine the purified conjugate to the precipitate.* To purify antibodies specific to the peptide, prepare an affinity column by immobilizing the peptide through the same functional group used to prepare the immunogen. The SulfoLink Coupling Gel (Product No. 20402) contains an activated gel support that will couple peptides via sulfhydryl groups. The peptide affinity column can then be used to specifically bind anti-peptide antibodies from serum, allowing antibodies against the carrier protein to flow through the column. Peptide-specific antibodies can then be eluted and recovered.。