Giant unilamellar vesicles (GUVs) constitute a cell-sized model membrane system that allowsdirect visualizationof particular membrane-related phenomena, such as domain formation, at the level of single vesicles using fluorescence microscopy-related techniques. Currently available protocols for the preparation of GUVs work only at very low salt concentrations, thus precluding experimentation under physiological conditions. In addition, the GUVs thus obtained lack membrane compositional asymmetry. Here we show how to prepare GUVs using a new protocol based on the electroformation method either from native membranes or organic lipid mixtures atphysiological ionic strength. Additionally, we describe methods to test whether membrane proteins and glycosphingolipids preserve their natural orientation after electroformation of GUVs composed of native membranes.
