Immunoprecipitation and Immunoblotting for Protein Interactions

Approximately 2 × 10⁷ of HEK-293 transfected cells were harvested after 48 hours of transfection and washed twice with ice-cold PBS. Cells were then lysed in 1 ml of modified RIPA lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA and 1% Triton X-100) supplemented with mini complete protease inhibitor cocktail (Roche Diagnostics, Laval, Quebec) for 30 min at 4 °C. Extracts were centrifuged at 14,000 rpm for 10 minutes at 4 °C to remove cell debris. 500 μg of total cell lysates were added to 100 μl of 50% slurry of anti-FLAG M2 affinity agarose beads (Sigma Aldrich, St. Louis, MO), pre-equilibrated with ice cold washing buffer (50 mM Tris-HCl pH 7.4 and 150 mM NaCl) and incubated overnight at 4 °C with continuous end-over-end rotation24 (link). Protein complexes were collected by centrifugation and washed four times in washing buffer and bound proteins were eluted with 100 μl of 3 × FLAG tag peptide at 150 μg/ml as recommended by the manufacturer (Sigma Aldrich, St. Louis, MO). Specific antibodies against DNAJB3 and JNK were then used to check for their interactions by immunoblotting.