This paper characterizes the caged-biotin−BSA system developed for selectively patterning biotinylated proteins into patterns on glass slides. Methyl α-nitropiperonyloxycarbonyl biotin, caged biotin, is coupled to a bovine serum albumin (BSA) carrier molecule using succinimide chemistry and then employed in a four-step patterning process: (1) caged-biotin−BSA compound is adsorbed onto a glass slide; (2) the slide is irradiated with 353-nm light through a chrome-on-glass photomask; (3) streptavidin is selectively bound to the irradiated regions; and (4) biotinylated protein is bound to the selectively immobilized streptavidin. A step-and-repeat scheme was used to demonstrate that streptavidin and then biotinylated BSA can be sequentially immobilized with reproducible feature density and little interfeature binding. Eight-minute irradiation of a mixed monolayer of 25% native BSA and 75% caged-biotin−BSA yielded the highest feature contrast, required the minimum use of reagent, and produced the least nonspe...