The basic principle for probing the transverse distribution of membrane lipids is simple. Lipids that are available for modification by enzymes or for covalent labeling in impermeable membrane vesicles are considered to be in the outer leaflet of the membrane bilayer, and those that are also accessible in permeable vesicles or only accessible in inside-out vesicles are considered to be in the inner leaflet of the membrane bilayer. However, the characteristics of the membrane preparation under investigation and the enzyme probe are critical in these experiments. The following criteria are important.
