Methods and Strategies to Quantify Phase Separation of Disordered Proteins

Phase separation has emerged as a new paradigm currently revolutionizing our understanding of cell biology and intracellular organization. Disordered protein domains have recently been demonstrated as integral drivers of phase separation into condensed liquids with emergent material properties. Using in vitro model systems employing purified protein components is necessary to interrogate the molecular mechanisms underlying phase separation; however, these systems pose many experimental challenges. In this chapter we describe general strategies for purifying, handling, imaging, and characterizing the phase behavior of disordered proteins. We further outline methods for the purification of the model P granule protein LAF-1, the construction of phase diagrams, and the quantification of liquid droplet fusion or coalescence.