The approach has been successfully applied for sodium and calcium binding to various membranes, but care must be taken to confirm that the simulation setup is sufficiently close to experiments, particularly in terms of molecular concentrations in the system and equilibration of the binding. While changing the hydration level with constant ion concentration did not affect the conclusions in previous sodium and calcium simulations, the situation is more complex for small molecules with stronger binding affinities. The concentration of these molecules substantially decreases in bulk water upon binding to the membrane.