Fluorescence-activated particle sorting for condensate purification.

IF 13.1 1区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS
Annie Munier Godebert, Dominique Weil, Adham Safieddine
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引用次数: 0

Abstract

Condensates are receiving increasing attention because of their ability to organize subcellular space. In eukaryotes, nuclear condensates include nucleoli and paraspeckles, and cytoplasmic ones include P-bodies (PBs) and stress granules. One approach to investigate condensate biology is through analyzing their protein and RNA content. However, purifying condensates remains a challenge because of their densities being similar to various other organelles, and the absence of protein markers accumulating exclusively in them. These limitations, combined with the generally low number of condensates per cell, necessitate new approaches to tackle their purification. Here, we present a protocol describing fluorescence-activated particle sorting (FAPS) for purifying condensates. In brief, FAPS involves fluorescently labeling condensates to identify and isolate them from other cellular components via sorting. In this Protocol, we focus on PB purification, quality control and downstream characterization of PB protein and RNA contents. Although originally developed to purify PBs from human cell lines, FAPS can be adapted to various condensates across model organisms. The procedure requires knowledge in basic cell culture, molecular biology and flow cytometry and access to a fluorescence-activated cell sorter with sufficient sensitivity. It requires ~25-30 d, including a hands-on period of 15 d, to complete. In summary, FAPS allows the characterization of the content of diverse condensates across cell types and organisms.

用于冷凝水净化的荧光活化颗粒分选。
凝聚体因其组织亚细胞空间的能力而受到越来越多的关注。在真核生物中,核凝聚物包括核仁和副斑,细胞质凝聚物包括p体和应力颗粒。研究凝析物生物学的一种方法是通过分析它们的蛋白质和RNA含量。然而,纯化凝聚体仍然是一个挑战,因为它们的密度与其他各种细胞器相似,并且缺乏专门积累在其中的蛋白质标记物。这些限制,加上每个电池的冷凝物数量普遍较低,需要新的方法来解决它们的净化问题。在这里,我们提出了一种描述荧光活化颗粒分选(FAPS)净化冷凝物的方案。简而言之,FAPS涉及荧光标记凝聚物,通过分选将其从其他细胞成分中识别和分离出来。在本议定书中,我们重点研究了PB的纯化、质量控制以及PB蛋白和RNA含量的下游特性。虽然最初是为了从人类细胞系中纯化PBs而开发的,但FAPS可以适用于模式生物的各种冷凝物。该程序需要基本的细胞培养知识,分子生物学和流式细胞术,并获得足够灵敏度的荧光激活细胞分选器。需要25-30天,包括15天的动手时间,才能完成。总之,FAPS允许表征不同类型的细胞和生物体凝聚物的含量。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Nature Protocols
Nature Protocols 生物-生化研究方法
CiteScore
29.10
自引率
0.70%
发文量
128
审稿时长
4 months
期刊介绍: Nature Protocols focuses on publishing protocols used to address significant biological and biomedical science research questions, including methods grounded in physics and chemistry with practical applications to biological problems. The journal caters to a primary audience of research scientists and, as such, exclusively publishes protocols with research applications. Protocols primarily aimed at influencing patient management and treatment decisions are not featured. The specific techniques covered encompass a wide range, including but not limited to: Biochemistry, Cell biology, Cell culture, Chemical modification, Computational biology, Developmental biology, Epigenomics, Genetic analysis, Genetic modification, Genomics, Imaging, Immunology, Isolation, purification, and separation, Lipidomics, Metabolomics, Microbiology, Model organisms, Nanotechnology, Neuroscience, Nucleic-acid-based molecular biology, Pharmacology, Plant biology, Protein analysis, Proteomics, Spectroscopy, Structural biology, Synthetic chemistry, Tissue culture, Toxicology, and Virology.
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