Protocol to analyze lipid asymmetry in the plasma membrane

The plasma membrane containing cholesterol exhibits phospholipid asymmetry, with phosphatidylcholine and sphingomyelin enriched in its outer leaflet and phosphatidylserine (PtdSer) and phosphatidylethanolamine (PtdEtn) on the cytoplasmic side. We herein describe steps for bacterial expression of recombinant proteins that bind to membrane lipids, followed by affinity purification. Using fluorescence-labeled phospholipid analogs, we further detail the assay to detect flippase activity, which maintains the single-sided distribution of PtdSer and PtdEtn, in mammalian cells. For complete details on the use and execution of this protocol, please refer to Segawa et al. (2021).1.