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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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