Most studies of the action of a peptidase on a supposed physiological substrate are performed in vitro. It may be, however, that in vivo peptidase and substrate do not meet, either because of a physical boundary, such as being in different intracellular (or extracellular) compartments, because inhibitors inactivate the peptidase, the cleavage sites are inaccessible because the substrate is bound to another protein, or the environment is unsuitable and the peptidase is not active.