Desthiobiotin-PEG3-NHS ester is a monocyclic, sulfur-free biotin analog characterized by a binding specificity to streptavidin that is nearly identical to that of biotin, although with a lower affinity (Kd = 10^11 compared to 10^15 M). Consequently, desthiobiotinylated proteins can be gently and specifically eluted from streptavidin affinity resins under conditions that involve competitive displacement with free biotin.