Middaugh C R, Vanin E F, Ji T H
Mol Cell Biochem. 1983;50(2):115-41. doi: 10.1007/BF00285638.
The complexity of cell membranes makes the resolution of their macromolecular topology one of the more challenging problems in modern molecular and cellular biochemistry. Despite the difficulties inherent in any such analysis, a surprisingly simple yet powerful approach exists that has consistently yielded valuable results. This method is chemical crosslinking, in which cell membranes are treated with crosslinking reagents (usually bifunctional) which produce covalent linkages between membrane components. The resultant complexes are usually then separated and identified by electrophoresis. This review is intended to provide a guide to the investigator who is unfamiliar with this approach. The overall strategy of crosslinking is discussed including selection of reagents, conditions to optimize crosslinking and the cleavage of crosslinked complexes to regenerate the original target for identification purposes. The crosslinking of biological membranes is then reviewed with special emphasis on recent advances including macromolecular photoaffinity labeling, kinetic analysis to probe symmetry properties and potential artifacts that may complicate interpretation of results. Examples of specific applications of crosslinking to membranes are presented in tabular form. The final portion of the review discusses the synthesis and properties of the most widely employed crosslinking reagents. Available reagents are summarized in a series of comprehensive tables. It is hoped that our discussion will provide the uninitiated investigator with sufficient information to ascertain the applicability of chemical crosslinking to particular areas of interest.
细胞膜的复杂性使得解析其大分子拓扑结构成为现代分子与细胞生物化学中更具挑战性的问题之一。尽管任何此类分析都存在固有困难，但有一种出人意料地简单却强大的方法，一直能产生有价值的结果。这种方法就是化学交联，即用交联试剂（通常是双功能的）处理细胞膜，这些试剂会在膜成分之间形成共价键。然后通常通过电泳分离并鉴定所得的复合物。这篇综述旨在为不熟悉这种方法的研究者提供指导。文中讨论了交联的总体策略，包括试剂的选择、优化交联的条件以及裂解交联复合物以再生用于鉴定目的的原始靶标。接着回顾了生物膜的交联，特别强调了近期的进展，包括大分子光亲和标记、用于探究对称性性质的动力学分析以及可能使结果解释复杂化的潜在假象。以表格形式展示了交联在膜上具体应用的实例。综述的最后部分讨论了最广泛使用的交联试剂的合成与性质。一系列综合表格总结了可用的试剂。希望我们的讨论能为新手研究者提供足够的信息，以确定化学交联在特定感兴趣领域的适用性。
