Couvillion Mary T, Collins Kathleen
Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA 94720-3200, USA.
Methods Cell Biol. 2012;109:347-55. doi: 10.1016/B978-0-12-385967-9.00012-8.
Epitope tagging is a powerful approach used to enable investigations of a cellular component by elucidating its localization, interaction partners, and/or activity targets. Successful tag-based affinity purification yields a mixture of the molecule of interest, associated proteins and nucleic acids, and nonspecific background proteins and nucleic acids, many of which can depend on details of the protocol for enrichment. This chapter provides guidelines and considerations for designing an affinity purification experiment, beginning with construction of a strain expressing a tagged subunit. Common biochemical methods for detecting protein, RNA, and DNA in Tetrahymena thermophila are also discussed.
表位标签是一种强大的方法，用于通过阐明细胞成分的定位、相互作用伙伴和/或活性靶点来研究该细胞成分。基于标签的成功亲和纯化会产生感兴趣分子、相关蛋白质和核酸以及非特异性背景蛋白质和核酸的混合物，其中许多可能取决于富集方案的细节。本章提供了设计亲和纯化实验的指南和注意事项，从构建表达带标签亚基的菌株开始。还讨论了嗜热四膜虫中检测蛋白质、RNA和DNA的常见生化方法。
