Production of a site- and phosphorylation state-specific antibody
Production of a site- and phosphorylation state-specific antibody
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DOI：
10.1038/nprot.2007.374
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发表时间：
2007-01-01
期刊：
影响因子：
14.8
通讯作者：
Inagaki, Masaki
中科院分区：
文献类型：
--
作者：
Goto, Hidemasa;Inagaki, Masaki
蛋白质磷酸化在细胞事件的各个方面起着重要作用。细胞内位点特异性磷酸化的可视化不仅对于分析磷酸化的时空分布，而且对于研究其生物学功能具有重要意义。现在，位点特异性和磷酸化状态特异性抗体被广泛用作这些分析的最有力工具。该方案详细描述了通过免疫对应于在靶位点磷酸化的蛋白质的合成磷酸肽来产生这种抗体的多克隆形式的方法。该方案也适用于生产其他类型的抗体，其特异性识别位点特异性修饰，如乙酰化、甲基化和蛋白水解。该方案可在2-3个月内完成。
Protein phosphorylation plays important roles in various aspects of cellular events. Visualization of site-specific phosphorylation in cells is of great importance not only to analyze spatial and temporal distribution but also to investigate biological function. Now, site- and phosphorylation state-specific antibodies are widely utilized as the most powerful tools for these analyses. This protocol details a method to produce the polyclonal version of such an antibody by immunizing a synthetic phosphopeptide corresponding to a protein phosphorylated at targeted site(s). This protocol is also applicable to the production of other types of antibodies, which specifically recognize the site- specific modification, such as acetylation, methylation and proteolysis. The protocol can be completed in 2-3 months.