Cholesterol is important for the formation of microdomains in supported lipid bilayers and is enriched in the liquidordered
phase. To understand the interactions leading to this enrichment, we developed an AFM-based single-lipid-extraction
(SLX) approach that enables us to determine the anchoring strength of cholesterol in the two phases of a phase-separated lipid
membrane. As expected, the forces necessary for extracting a single cholesterol molecule from liquid-ordered phases are significantly
higher than for extracting it from the liquid-disordered phases. Interestingly, application of the Bell model shows two
energy barriers that correlate with the head and full length of the cholesterol molecule. The resulting lifetimes for complete
extraction are 90 s and 11 s in the liquid-ordered and liquid-disordered phases, respectively. Molecular dynamics simulations
of the very same experiment show similar force profiles and indicate that the stabilization of cholesterol in the liquid-ordered
phase is mainly due to nonpolar contacts.