Metabolic labeling, biotin conjugation, and streptavidin pull-down of nascent proteins

After 1-hour incubation in methionine free medium (Thermo Fisher Scientific 21013024), nascent proteins were labeled for 2 hours with 1 mM AHA (L-azidohomoalanine, Sigma 900892). AHA was washed away, and cells either collected for protein extracts or fed with complete differentiation medium then collected for protein extracts at the indicated time points. The cells were dissociated using accutase and rinsed in PBS, and the pellets were frozen on dry ice and stored at −80°C until needed. To prepare protein extracts, cells were lysed in 50 mM Tris pH 8, 1% SDS, 250 U/ml benzonase (Pierce 88700), and one tablet per 3.5 ml of lysis buffer protease inhibitors (Roche 04693159001). Protein content of the cell lysates was measured using the BCA protein assay kit (Pierce 23225). One milligram of total protein per sample was labeled using biotin alkyne (Invitrogen B10185) and Click-it technology (Click it Protein Reaction Buffer kit, Invitrogen 10276). Labeled protein extracts were purified by desalting columns Zeba 7-kDa MWCO (Thermo Fisher Scientific 89892) and mixed with washed magnetic streptavidin beads (400 μl of slurry per sample, Thermo Fisher Scientific 65602) for 16 hours at 4°C. The beads were washed four times with 0.1% SDS, 0.1% BSA PBS, once with 0.1% SDS, 0.1% BSA PBS, and biotinylated proteins were recovered by heating at 92°C for 10 min in 2X Laemmli buffer.