Protocol to compare relative protein-liposome binding affinity using a fluorescence microscopy-based approach

Centrifugation-based protein-liposome assays are unsuitable for spontaneously precipitating proteins and have limited quantification capabilities. Here, we present a protocol to compare relative protein-liposome binding affinity using a fluorescence microscopy-based approach. We described steps for fluorescent liposome preparation, fission yeast protein extraction, liposome binding assay, and confocal imaging. We also provided analysis methods for different setups. Although this protocol is established for fission yeast, it can be applied to most non-transmembrane proteins obtained from various in vivo and in vitro systems. For complete details on the use and execution of this protocol, please refer to Hoh et al.¹.