Biochemical Purification of Native Immune Protein Complexes
Biochemical Purification of Native Immune Protein Complexes
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DOI：
10.1007/978-1-61737-998-7_4
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发表时间：
2011-01-01
期刊：
影响因子：
--
通讯作者：
Coaker, Gitta
中科院分区：
文献类型：
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作者：
Elmore, James M.;Coaker, Gitta
蛋白质复合物纯化是识别植物先天免疫中新参与者的有效方法。然而，在自然环境中识别相互作用的蛋白质伙伴一直是研究人员面临的挑战。在本章中，我们描述了一种使用纯化抗体从野生型组织中分离天然蛋白质复合物的免疫亲和层析方法。除了免疫共沉淀方案之外，我们还详细介绍了抗体纯化和固定步骤。此外，还描述了一种制备用于质谱分析的蛋白质样品的方法。这种简单的方案已用于分离和鉴定拟南芥免疫相关蛋白复合物的新成分。
Protein complex purification represents a powerful approach to identify novel players in plant innate immunity. However, the identification of interacting protein partners within a natural context has been a challenge for researchers. In this chapter, we describe a method of immunoaffinity chromatography using purified, antibodies to isolate native protein complexes from wild-type tissue. We detail the antibody purification and immobilization steps in addition to the co-immunoprecipitation protocol. In addition, a method to prepare protein samples for mass spectroscopy analysis is described. This straightforward protocol has been used to isolate and identify novel components of Arabidopsis immunity-associated protein complexes.