融合蛋白
串联亲和纯化
蛋白质标签
靶蛋白
生物化学
计算生物学
生物素化
荧光团
化学
表位
分子生物学
生物
亲和层析
抗体
重组DNA
酶
荧光
遗传学
基因
物理
量子力学
作者
Ronald T. Raines,Michael L. McCormick,Thomas R. Van Oosbree,Robert C. Mierendorf
出处
期刊:Methods in Enzymology
日期:2000-01-01
卷期号:: 362-376
被引量:69
标识
DOI:10.1016/s0076-6879(00)26065-5
摘要
This chapter discusses protein purification using S. tag fusion system. The S. Tag fusion system uniquely combines small tag size, antibody like ligand-binding specificity, and the ability to confer an easily measured enzymatic activity to fusion proteins. The S-protein ligand is also small, relatively inexpensive, and can be used in a variety of formats to enable many applications with a single tagging system. Virtually every technique used with antibodies and their epitope tags can be applied to the S-protein: S. Tag interaction, including the blotting and purification methods, fusion protein immobilization, affinity capture of interacting molecules, and the use of fluorophore-labeled S-protein for in situ affinity localization and cell sorting. The reconstitution of enzymatic activity by the simple addition of S-protein to any S.Tag fusion protein provides the platform for the development of novel assays. In particular, the discovery of a hypersensitive fluorogenic substrate for RNase A makes assays of S. Tag fusion proteins amenable to automation, and thus useful in a variety of high-throughput screening applications.
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