Protocols for in vitro analysis of liquid-liquid phase separation of proteins

Liquid-liquid phase separation (LLPS) plays a central role in the organization of biomolecular condensates that regulate diverse cellular processes. In vitro LLPS assays provide a versatile platform for studying the molecular determinants that govern condensate formation, morphology, dynamics, and modulation. This chapter presents a detailed protocol for performing in vitro LLPS assays, particularly using polyethylene glycol (PEG) and sodium salt as key modulators of phase behavior. The described procedure enables systematic evaluation of protein condensation under defined physicochemical conditions, thereby facilitating insights into the interplay among macromolecular crowding, ionic strength, and protein concentration. The protocol includes guidelines for sample preparation, phase separation induction, microscopy-based visualization, and quantitative analysis of condensate properties. Collectively, this toolkit provides a robust framework for studying the biophysical principles underlying LLPS and for assessing the effect of environmental or molecular perturbations on protein phase behavior.