Biochemical Reconstitution and FRAP Analysis of Membrane-Associated Condensates on Supported Lipid Bilayers

Plasma membrane-associated condensates driven by liquid-liquid phase separation represent a novel mechanism of receptor-mediated signaling transduction, serving as mesoscale platforms that concentrate signaling molecules and modulate reaction kinetics. Condensate formation is a highly dynamic process that occurs within seconds to minutes following receptor activation. Here, we present methods for de novo reconstituting liquid-like condensates on supported lipid bilayers and assessing the condensate fluidity using fluorescence recovery after photobleaching (FRAP). This protocol encompasses supported lipid bilayer preparation, condensation imaging, and FRAP analysis using total internal reflection fluorescence (TIRF) microscopy. Supported lipid bilayers provide a membrane-mimicking environment for receptor signaling cascades, offering mechanistic insights into protein-protein and lipid-protein interactions amid micron-scale condensates. The protocol can also be adapted to study condensates associated with the internal membranes of the Golgi apparatus, mitochondria, and other organelles. Key features • Real-time imaging of condensate formation and FRAP analysis using TIRF microscopy reveals a spatiotemporal profile of signaling transduction. • Supported lipid bilayers provide a fluidic membrane environment that is critical for the biochemical reconstitution of condensates at physiological protein concentrations. • The lipid and protein components within the reconstituted condensate system can be readily manipulated to accommodate specific experimental objectives and assay designs.