Singh Chingakham Ranjit, Asano Katsura
Molecular, Cellular, and Developmental Biology Program, Division of Biology, Kansas State University, Manhattan, Kansas, USA.
Methods Enzymol. 2007;429:139-61. doi: 10.1016/S0076-6879(07)29007-X.
This chapter aims to describe methods to identify and characterize protein-protein interactions that were developed during our studies on translation initiation factor complexes. Methods include the two-hybrid assay, the GST pull-down assay, and the coimmunoprecipitation (co-IP) assay. The two-hybrid assay provides for a convenient start to find the minimal interaction domains, which generally produce well-behaved recombinant proteins suited for various in vitro interaction assays. Emphasis is placed on demonstrating physiological relevance of identified interactions. The effective strategy is to find mutations that reduce the interaction by genetic or site-directed mutational approaches and obtain correlations between their effects in vitro (GST pull down) and effects in vivo (co-IP).
本章旨在描述我们在对翻译起始因子复合物的研究过程中开发的用于鉴定和表征蛋白质-蛋白质相互作用的方法。这些方法包括双杂交测定、GST下拉测定和免疫共沉淀（co-IP）测定。双杂交测定为寻找最小相互作用结构域提供了便利的开端，该结构域通常会产生适用于各种体外相互作用测定的性能良好的重组蛋白。重点在于证明所鉴定相互作用的生理相关性。有效的策略是通过遗传或定点突变方法找到能降低相互作用的突变，并获得它们在体外（GST下拉）和体内（co-IP）效应之间的相关性。
