Biotin-Phenol Labeling of Cultured Cells

Stable cells were grown in four T75 flasks. Cells were induced with doxycycline at 60–80% confluence. After 18–24 h, the medium in each flask was changed to 7.5 mL of fresh growth medium containing 250 µM desthiobiotin-phenol or biotin-phenol. All DBP and BP parallel experiments were performed simultaneously with the same protocol. The flasks were incubated at 37 °C under 5% CO₂ for 30 min according to previously published protocols. Afterward, 750 μL of 10 mM H₂O₂ (diluted from 30% H₂O₂, Sigma Aldrich H1009) was added to each flask for a final concentration of 1 mM H₂O₂, and the flasks were gently agitated for 1 min at room temperature. The reaction was then quenched by adding 7.5 mL of DPBS containing 10 mM Trolox, 20 mM sodium azide, and 20 mM sodium ascorbate to each flask. Then, the solution was removed, and the cells were washed three times with a cold quenching solution (DPBS containing 5 mM Trolox, 10 mM sodium azide, and 10 mM sodium ascorbate). Cells were detached using of cold quenching solution and centrifuged at 1,500×g for 5 min at 4 °C. Cells were resuspended with fresh cold quenching solution and centrifuged again.