Cell Lysis and Protein Normalization

Cell pellets were resuspended in lysis buffer containing 100 mM Hepes pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 2 mM PMSF, and 1× complete EDTA-free protease inhibitor cocktail (Roche, Millipore Sigma). Lysates were mixed thoroughly by pipetting up and down with 1-mL Eppendorf pipette tips and then allowed to sit on ice for 30 min. Cellular debris was removed by centrifugation at 14,000g for 30 min at 4 °C. Protein concentrations were determined by bicinchoninic acid (BCA) assay (Bio-Rad), and cell lysates were diluted with lysis buffer to normalize final protein concentrations, typically to ∼3 mg/ml. Portions of the samples to be reacted with biotin-azide for affinity purification using the original OPP-ID protocol (16 (link)) were precleared by overnight incubation with streptavidin agarose resin (Thermo Scientific) at 4 °C with slow rotation prior to click chemistry.