Affinity Purification of Protein Complexes

Cells cultured in trays (Thermo, cat#166508) were cross-linked, washed with cold PBS, scraped off, and snap-frozen. The cell pellets were resuspended in extraction buffer (25 mM Tris, pH 7.5, 100 mM NaCl, 5 mM MgCl₂, 0.2% NP-40, 10% glycerol, EDTA-free protease inhibitor tablet, and 1 mM PMSF). After brief sonication (2 × 10 s) and centrifugation (1700 × g, 2 min), the pellets were resuspended in extraction buffer supplemented with 90 U/ml benzonase (Millipore, cat#70664) and rotated at 4 °C for 1 h. Ten volumes of RIPA buffer I (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, and 1 mM PMSF) were added, followed by sonication (6 × 10 s). The supernatants were clarified by centrifugation at 13,000 rpm for 10 min and rotated overnight at 4 °C with 100 µl antibody-coupled beads (Herzog and Peters, 2005 (link)). Beads were then washed twice with RIPA buffer I, twice with RIPA buffer II (50 mM Tris, pH 7.5, 500 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, and 1 mM PMSF), twice with RIPA buffer III (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, and 1 mM PMSF), and twice with 150 mM NaCl. Bound protein complexes were eluted twice with 50 µl of 0.1 M glycine (pH 2.0), pooled, neutralized with 7 µl of 1.5 M Tris-HCl (pH 9.2), and used for silver staining, western blotting, or MS.