This work examines the molecular influence of the local environment on the apparent properties of immobilized proteins. Using the surface force apparatus and cytochrome b5 immobilized to different supports, we measured directly the range and magnitude of molecular forces originating from the underlying matrix. in particular, we used as supports oriented streptavidin monolayers, charged maleimide-functionalized phospholipid bilayers, and neutral maleimide-functionalized lipid bilayers. The relative impact of the underlying surface forces on the apparent electrostatic properties of the bound proteins was evaluated, as was the efficacy of different methods such as electrostatic screening and charge neutralization on the ability to minimize their influence. These background forces not only mask structural features of the proteins that may direct ligand trajectories to their binding sites, but they also alter the efficiencies of protein coupling to the solid support. The results presented provide valuable information for the design of immobilization matrices.