Production of antisera using fusion proteins.
Production of antisera using fusion proteins.
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使用融合蛋白生产抗血清。
DOI：
10.1002/0471142301.ns0507s40
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发表时间：
2007
影响因子：
--
通讯作者：
Levey,AllanI
中科院分区：
文献类型：
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作者：
Dodson,SaraE;Heilman,CraigJ;Kahn,RichardA;Levey,AllanI
本单元详细介绍了使用细菌产生的融合蛋白来生产抗血清，从而可以大规模生成针对特定目标表位的亲和纯化抗体。使用含有多组氨酸 (His) 或谷胱甘肽-S-转移酶 (GST) 标签的 pET 载体构建细菌表达质粒作为融合蛋白方法的典型示例。本单元提供的基本协议描述：（1）E 的转换。用于高产生产可溶性融合蛋白的大肠杆菌，(2) 使用螯合镍或谷胱甘肽亲和层析（分别针对 His 和 GST 标记的融合蛋白）纯化用于免疫的可溶性融合蛋白，(3) 用纯化的融合蛋白对兔子进行免疫并收集抗血清，以及 (4) 使用 免疫印迹技术。支持方案描述了用于动物免疫的 His 标记的不溶性融合蛋白的纯化，以及使用可溶性融合蛋白纯化抗体的亲和柱的构建和使用。协议。神经科学。 40：5.7.1‐5.7.26。 © 约翰·威利父子公司 (John Wiley & Sons, Inc.)
This unit details the use of bacterially produced fusion proteins for the production of antisera, allowing for the large‐scale generation of affinity‐purified antibodies to specific, targeted epitopes. The use of pET vectors containing a polyhistidine (His) or glutathione‐S‐transferase (GST) tag to construct bacterial expression plasmids are provided as prototypical examples of fusion protein methodology. The basic protocols provided in this unit describe: (1) transformation ofE. colifor high‐yield production of soluble fusion protein, (2) purification of soluble fusion proteins for use in immunization using chelated nickel or glutathione affinity chromatography (for His‐ and GST‐tagged fusion proteins, respectively), (3) immunization of rabbits with purified fusion protein and collection of antisera, and (4) characterization of antisera for antibody specificity using immunoblotting techniques. Support protocols describe the purification of His‐tagged insoluble fusion proteins for animal immunization and the construction and use of affinity columns for purifying antibodies using soluble fusion proteins.Curr. Protoc. Neurosci. 40:5.7.1‐5.7.26. © by John Wiley & Sons, Inc.