Protocol to study membrane-associated protein phase separation using protein-lipid blot overlay assays and giant unilamellar vesicles in vitro

Membrane-associated protein phase separation plays a critical role in cellular signaling. Here, we present a protocol for studying protein-lipid interactions and membrane-associated phase separation using protein-lipid blot overlay assays and giant unilamellar vesicles (GUVs). We describe steps for performing protein-lipid overlay assays to screen for lipid-binding specificity, followed by procedures for GUV preparation and protein-binding assays to visualize phase separation on membrane surfaces in vitro. This protocol applies to any protein capable of both phase separation and membrane binding. For complete details on the use and execution of this protocol, please refer to Xie et al.