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The regulation of protein translation has a role in several aspects of biology; tools to study it are therefore in demand. Salic and colleagues report that an alkyne analog of the aminonucleoside antibiotic puromycin can be incorporated into translating polypeptide chains and can be used to label them for fluorescence detection or affinity purification. Like puromycin, the O-propargyl puromycin analog blocks protein synthesis by generating covalent conjugates to the growing nascent chain; the terminal alkyne is then available for labeling via click chemistry. The approach is rapid, does not require methionine-free medium and is compatible with monitoring protein synthesis in cells and in whole organisms.