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Protocol | Open Access

Protocol for analyzing protein liquid–liquid phase separation

Zheng Wang1Gangming Zhang1Hong Zhang1,2( )
National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China
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Graphical Abstract


Numerous cellular functions occur in spatially and temporally confined regions. Recent studies have shown that membrane-less organelles and compartments in the cell are assembled via liquid–liquid phase separation (LLPS). In vitro LLPS assays using recombinant expressed and purified proteins are necessary for us to further understand how the assembly of phase-separated compartments is regulated in cells. However, uniform standards and protocols are lacking for these in vitro studies. Here, we describe a step-by-step protocol commonly used to investigate in vitro LLPS using purified proteins. This protocol includes expression and purification of the studied proteins, induction of LLPS of the purified proteins, and studies of the biophysical properties of the liquid droplets formed by LLPS. These protocols can be easily followed by researchers to investigate the LLPS behaviors of proteins of interest.



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Biophysics Reports
Pages 1-9
Cite this article:
Wang Z, Zhang G, Zhang H. Protocol for analyzing protein liquid–liquid phase separation. Biophysics Reports, 2019, 5(1): 1-9.











Received: 29 September 2018
Accepted: 16 October 2018
Published: 05 December 2018
© The Author(s) 2018

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (, which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.