生物物理学
体外
化学
突变体
色谱法
细胞生物学
生物
材料科学
生物化学
基因
作者
Peter Raymond-Smiedy,Barrington Bucknor,Yiran Yang,Tongyin Zheng,Carlos Castañeda
出处
期刊:Methods in molecular biology
日期:2022-10-31
卷期号:: 515-541
被引量:4
标识
DOI:10.1007/978-1-0716-2597-2_32
摘要
Liquid-liquid phase separation (LLPS) is hypothesized to be the underlying mechanism for how membraneless organelles or biomolecular condensates form inside both prokaryotic and eukaryotic cells. Protein LLPS is a biophysical process during which proteins demix from homogeneous solution to form protein-dense droplets with liquid-like properties. Disruptions to LLPS, such as changes to material properties of condensates or physicochemical parameters for LLPS onset, are implicated in neurodegenerative diseases and cancer. Therefore, it is essential to determine the physicochemical parameters that promote protein LLPS. Here, we present our UV-Vis spectrophotometric turbidity assay to characterize the temperature and concentration dependence of LLPS for UBQLN2, a protein that undergoes LLPS via homotypic interactions in vitro and forms stress-induced condensates in cells. Mutations in UBQLN2 cause amyotrophic lateral sclerosis (ALS) and disrupt UBQLN2 LLPS. We present a detailed expression and purification protocol for a C-terminal construct of UBQLN2 and how we use microscopy to image UBQLN2 LLPS. We use our UV-Vis assay to construct temperature-concentration phase diagrams for wild-type and mutant UBQLN2 constructs to determine the effects of domain deletions and/or mutations on UBQLN2 phase separation.
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