A protocol for mimicking lipid-mediated phase separation on the membrane using giant unilamellar vesicles

In addition to creating a physical boundary, cell membranes provide critical platforms for many biological processes. Due to the complexity of the membrane composition, it is a challenging task to study the functions of membrane-associated proteins. Here, we present a general protocol by which we use giant unilamellar vesicles (GUV) as a tool to study AGO proteins’ behavior on the membrane. We provide procedures to prepare fluorescent-labeled protein and GUVs and describe the protein phase separation in 3D time-lapse images.