光漂白后的荧光恢复
核糖核酸
液态液体
体内
荧光显微镜
光漂白
体外
化学
生物物理学
细胞生物学
色谱法
生物
荧光
生物化学
物理
遗传学
膜
量子力学
基因
作者
Stephanie Heinrich,Maria Hondele
出处
期刊:Methods in molecular biology
日期:2022-01-01
卷期号:: 307-333
被引量:4
标识
DOI:10.1007/978-1-0716-2521-7_18
摘要
Biomolecular condensates and the concept of liquid-liquid phase separation (LLPS) have transformed cell biology in recent years. Condensates organize cellular content and compartmentalize biochemical reactions, in particular many processes involving RNA. This protocol is aimed at readers new to the LLPS field who want to test their protein or cellular structure of interest. We describe the basic principles of liquid-liquid phase separation, and outline initial approaches-both in vitro and in yeast cells-for the characterization of a candidate cellular condensate. First, we focus on strategies to purify phase-separating proteins and to reconstitute condensates from recombinant proteins in vitro for observation by light microscopy. Second, we describe in vivo experiments (including fluorescence recovery after photobleaching (FRAP) microscopy and 1,6-Hexanediol treatment) to test whether a subcellular structure displays liquid-like behavior in cells.
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