Quantifying Nascent Protein Synthesis

Metabolic labeling of nascent proteins with OP-puro was performed as previously described (Iwasaki et al., 2019 (link)). Cells were treated with 20 µM OP-puro and incubated at 37°C for 30 min in a CO₂ incubator. After washing with PBS, cells were lysed with buffer containing 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl₂, and 1% Triton X-100. Nascent polypeptides were labeled with IRdye800CW Azide (LI-COR Biosciences) with a Click-iT Cell Reaction Buffer Kit (Thermo Fisher Scientific). After free dye was removed by a G-25 column (Cytiva), labeled polypeptides were separated by SDS-PAGE. The gel was imaged by Odyssey CLx (LI-COR Biosciences) for the detection of nascent peptides with infrared at 800 nm. Then, total proteins were stained with CBB (FUJIFILM Wako Chemicals) and imaged with an infrared 700 nm signal. The gel area ranging from 17 kDa to 280 kDa was quantified using Image Studio (version 5.2, LI-COR Biosciences), and the nascent peptide signal was normalized to the total protein signal.