Immunoprecipitation and Affinity Purification

Cells were lysed in lysis buffer (25 mM Tris pH 8.0, 150 mM NaCl, 10% glycerol, 1 mM EDTA, 1 mM EGTA, 1 mM 1,4-Dithiothreitol (DTT) and 0.1% NP-40) supplemented with protease (Complete ULTRA, Roche) and phosphatase inhibitors (PhosSTOP, Roche). The insoluble fraction was removed by centrifugation (20,000 x g x 15 min at 4°C). Immunoprecipitations and affinity precipitations were carried out using FLAG-M2 agarose (Sigma-Aldrich) or anti-HA Affinity Matrix (Roche) at 4°C for 1 hr. Beads were washed once in lysis buffer before washing with CSK buffer (10 mM PIPES, pH 7.0, 100 mM NaCl, 300 mM sucrose, 1 mM EGTA, 3 mM MgCl₂, 0.1% Triton X-100) containing 1 U/mL TurboNuclease (Accelagen) for 15 min at RT. Beads were extensively washed in lysis buffer and elution was carried out with 3 × FLAG peptide (Sigma-Aldrich) or HA peptide (Roche). Immunoblotting was performed as previously described (Pagan et al., 2015 (link)).