{"title":"Fluorescence-activated particle sorting for condensate purification.","authors":"Annie Munier Godebert, Dominique Weil, Adham Safieddine","doi":"10.1038/s41596-025-01216-x","DOIUrl":null,"url":null,"abstract":"<p><p>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.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1000,"publicationDate":"2025-07-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Nature Protocols","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1038/s41596-025-01216-x","RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
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.
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.