Cross-linking of flag Dynabeads Protein G

To cross-link the FLAG M2 antibody to magnetic beads, 25 μl of Protein G Dynabead slurry (Thermo Fisher Scientific) was washed three times with 0.05% Tween 20/PBS and then incubated with 4 μg of FLAG M2 monoclonal antibody (Sigma-Aldrich) for 2 hours at 4°C. Beads were washed in 0.2 M triethanolamine (TAE) in PBS, incubated with 20 mM dimethyl pimelimidate (DMP; Sigma-Aldrich) in 0.2 M TAE (pH 8.2) for 30 min at room temperature, quenched with 50 mM tris-HCl (pH 7.5), and washed again three times with 0.05% Tween 20/PBS.