In Vitro Transition Temperature Measurement of Phase-Separating Proteins by Microscopy

Intracellular compartmentalization through liquid-liquid phase separation is an emerging organizing principle of cell biology. These compartments, such as the nucleolus and stress granules, are collectively known as membraneless organelles or biomolecular condensates. In vitro studies of many protein components of biomolecular condensates, such as the intrinsically disordered regions of Ddx4, FUS, and Laf-1 proteins, have revealed much about the driving forces of the phase separation process. A common approach is to investigate how the temperature at which a protein solution forms condensates-the transition temperature-responds to changes in the solution composition. We describe a method to measure the in vitro transition temperature of a sub-10 μL sample of a phase-separating solution using transmitted light microscopy. Through monitoring changes in transition temperature with solution conditions, this approach allows the impact of additional biomolecules and additives to be quantitatively assessed and permits the construction of phase diagrams.