Protein g coated magnetic beads

Cells 10 × 10⁶ were grown to confluence in 150 mm plates, washed three times in PBS buffer and 2 ml of lysis buffer (20 mM Tris-HCl pH 7.8, 250 mM sucrose, 1 mM MgCl₂, and 1 mM CaCl₂) supplemented with protease (1 mM PMSF, 1 µg/ml leupeptin and aprotinin) and phosphatase (50 mM sodium fluoride, 2 mM sodium orthovanadate, 10 mM sodium pyrophosphate) inhibitors. Cells kept for 20 min over ice were scraped and collected in a final volume of 5 ml. The cell suspension was transferred to a 35 -ml nitrogen cavitation bomb for 30 min in 400-psi nitrogen pressure on ice. Intact cells, large debris and nucleus were removed by centrifugation at 1 000 g for 10 min at 4°C. Lysates were incubated overnight at 4 °C under agitation with 8 µg of PDIA1 antibody, followed by incubation with 70 μl of protein G-coated magnetic beads (ref. 28-9513-79 from GE Health Care) for 4 h at 4 °C. Beads were successively washed in sucrose buffer to remove contaminating material, ressuspended in modified FLAG lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 and 1% CHAPS) supplemented with a protease and phosphatase inhibitors. After 1 h of incubation at room temperature, Laemmli sample buffer was added and incubated at room temperature for additional 1 h. KRas was detected by immunoblot.