Determination of Protein Phase Diagrams by Centrifugation

Liquid-liquid phase separation (LLPS) underlies the formation of biomolecular condensates, i.e., membrane-less compartments in cells that carry out functions related to RNA metabolism, stress adaptation, transport, or signaling. Examples of such biomolecular condensates are the nucleolus, nuclear speckles, promyelocytic leukemia protein (PML) bodies and paraspeckles in the nucleus, and stress granules and P bodies in the cytoplasm. Other structures in cells that are not typically viewed as bona fide compartments also seem to be formed via LLPS as recently elucidated, including heterochromatin, super-enhancers, and membrane receptor clusters. Key protein and/or RNA components of these biomolecular condensates form a scaffold via LLPS. Other constituents incorporate into this scaffold as clients. To understand the sequence features and interactions that mediate biomolecular condensate formation in cells, it is useful to quantify phase separation of pure components in vitro. Microscopy and turbidity measurements can be used to determine the concentration of a protein above which it phase separates, the so-called saturation concentration. Here, we describe experiments for the determination of full coexistence lines of phase-separating proteins by centrifugation. Coexistence lines are reconstructed from coexisting light and dense phase concentrations of the protein, and we present them as so-called phase diagrams. Phase diagrams allow the quantitative comparison of phase separation for proteins and their mutants under different conditions. They are thus important for our nuanced understanding of the driving forces underlying liquid-liquid phase separation in vitro. Such results have direct applicability for understanding phase separation-driven compartmentalization of cells.