Methods to visualize, track, measure and perturb proteins in living cells are central to biomedicine's efforts to characterize and understand the spatial and temporal underpinnings of life inside cells. Although fluorescent proteins have revolutionized such studies, they have shortcomings, which have spurred the creation of alternative approaches to chemically label proteins in live cells. In this review we highlight research questions that can be addressed using site-specific chemical labeling and present a comparison of the various labeling techniques that have been developed. We also provide a 'roadmap' for selection of appropriate labeling techniques(s) and outline generalized strategies to validate and troubleshoot chemical labeling experiments.
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