FRAP and FRET Investigation of α-Synuclein Fibrillization via Liquid-Liquid Phase Separation In Vitro and in HeLa Cells

Liquid-liquid phase separation (LLPS) acts as an important biological phenomenon in membraneless organelle formation. These phase-separated bodies can also act as nucleation centers for disease-associated amyloid formation. Fluorescence recovery after photobleaching (FRAP) is a crucial technique to analyze the material property (liquid or solid) of protein LLPS. On the other hand, Förster resonance energy transfer (FRET) is used to understand the domain-specific involvement (intermolecular interactions) of protein molecules inside the phase-separated droplets. In this protocol, we delineate mechanisms of liquid-to-solid transition of α-synuclein LLPS by using in vitro and in cell FRAP as well as in vitro FRET techniques.