Nadeau Owen W, Carlson Gerald M
CSH Protoc. 2007 Apr 1;2007:pdb.prot4634. doi: 10.1101/pdb.prot4634.
INTRODUCTIONThis protocol describes a method for chemical cross-linking of proteins using formaldehyde. With the exception of zero-length cross-linkers, formaldehyde has the shortest cross-linking span (~2-3 Å) of any cross-linking reagent, thus making it an ideal tool for detecting specific protein-protein interactions with great confidence. Despite its simple chemical structure (CH(2)O), formaldehyde's single carbonyl group functions essentially as a homobifunctional reagent and is capable of conjugating targets through two different chemical pathways. In Mannich-type reactions, which typically require elevated temperatures (37°C or above) for a period of 2-24 hours for acceptable yields, formaldehyde condenses with amines (1°, 2°, or salts of ammonia) and compounds with active hydrogens to form stable cross-links. Formaldehyde also reacts with amines to form highly reactive immonium cations that are reactive toward protein-containing nucleophiles, including sulfhydryls, amines, phenols, and imidazoles. The latter reaction is more rapid than the former and is generally the more predominant in typical protein cross-linking reactions at ambient temperatures.
引言
本方案描述了一种使用甲醛对蛋白质进行化学交联的方法。除零长度交联剂外，甲醛是所有交联试剂中交联跨度最短的（约2 - 3埃），因此使其成为一种能够高度可靠地检测特定蛋白质 - 蛋白质相互作用的理想工具。尽管甲醛的化学结构简单（CH₂O），但其单个羰基基本上起同双功能试剂的作用，能够通过两种不同的化学途径结合靶标。在曼尼希型反应中，通常需要在高温（37°C或更高）下反应2 - 24小时才能获得可接受的产率，甲醛与胺（伯胺、仲胺或铵盐）以及具有活性氢的化合物缩合形成稳定的交联。甲醛还与胺反应形成高反应性的亚胺阳离子，这些阳离子对含蛋白质的亲核试剂具有反应性，包括巯基、胺、酚和咪唑。后一种反应比前一种反应更快，并且在环境温度下的典型蛋白质交联反应中通常更占主导地位。
