Labeling Nascent Polypeptides with Biotin-Puromycin

We labeled translating nascent polypeptides as described (15 (link)). In brief, ribosomes (4 μg of ribosomal RNA) were incubated with or without 2 μM of biotin-conjugated puromycin (Jena Bioscience) in a buffer containing 10 mM Tris (pH 7,4), 400 mM KCl and 3 mM MgCl_2. After 90 min at 37°C the reaction was stopped by adding Laemmli Buffer. For treatments, ribosomes were pre-incubated with either 0.1 μg/μl RNAse A or 50 mM EDTA for 15 min at 30°C. The reaction products were analyzed by Western blot using streptavidin-conjugated HRP (Abcam). For pull-down assays, after labeling, the reaction was dialyzed against a buffer containing 10 mM Tris (pH 7,4), 300 mM KCl and 3 mM MgCl_2. Then, streptavidin conjugated agarose beads (Thermo Scientific) were added, incubated for 90 min and washed with the dialysis buffer containing 0.01% NP40. The reaction products were analyzed by Western blot.