Lutter L C, Kurland C G
Mol Cell Biochem. 1975 May 30;7(2):105-16. doi: 10.1007/BF01792077.
Experimental procedures developed for the identification of near-neighboring proteins in a complicated organelle are described in this paper. The specific system studied here is the ribosome of Escherichia coli; however, the techniques and reagents described should have a wide range of applications. We have used three kinds of cross-linking agents: noncleavable maleimide reagents which react with free SH groups; noncleavable diimidoesters which react with free amino groups, and cleavable diazide agents that also react with free amino groups. The combination of immunological and isotopic labelling techniques used to identify proteins cross-linked by noncleavable reagents, as well as the special electrophoretic techniques used with the cleavable reagents are described. We show that the neighborhoods detected by the crosslink technology are functionally meaningful ones, and also discuss the ambiguities inherent in the structural interpretation of such protein neighborhoods.
本文描述了用于鉴定复杂细胞器中近邻蛋白质的实验程序。这里研究的特定系统是大肠杆菌核糖体；然而，所描述的技术和试剂应该有广泛的应用。我们使用了三种交联剂：与游离SH基团反应的不可裂解马来酰亚胺试剂；与游离氨基反应的不可裂解二亚胺酯，以及也与游离氨基反应的可裂解二叠氮试剂。描述了用于鉴定由不可裂解试剂交联的蛋白质的免疫和同位素标记技术的组合，以及与可裂解试剂一起使用的特殊电泳技术。我们表明，通过交联技术检测到的邻域具有功能意义，并讨论了此类蛋白质邻域结构解释中固有的模糊性。
