Pierce protein g agarose beads

Throughout this work, all immunoprecipitations were preceded by an in vitro antibody agarose bead crosslinking step, to prevent excessively abundant peptides from the antibodies which can generate unwanted interferences. Antibodies were incubated with Pierce Protein G Agarose beads (20398; Thermo Fisher Scientific) at a 1:20 ratio (5 μl of antibody for 100 μl of beads) for 1 h rocking. Antibody-bound beads (henceforth referred to simply as beads) were then washed with sodium tetraborate decahydrate (S9640; Sigma-Aldrich), 0.1 M, pH 9, and resuspended again in sodium tetraborate decahydrate with dimethyl pimelimidate (DMP) (21667; Thermo Fisher Scientific) at the final concentration of 20 mM. The DMP and beads were mixed for 30 min at RT, after which the reaction was terminated by washing the beads once with ethanolamine (E0135; Sigma-Aldrich) 0.2 M pH 8 and three times with PBS. Finally, beads were resuspended in PBS with 0.02% (wt/vol) sodium azide (S2002; Sigma-Aldrich) and stored at 4°C until use (no longer than 48 h).

Twenty-four hours prior to transfection, 1 x 10⁶ HEK293T cells or 4 × 10⁶ HCT116 control or METTL11A KO cells were plated in 10 cm tissue culture dishes. HEK293T cells were calcium phosphate transfected with 1 μg each of appropriate constructs, and HCT116 cells were transfected with 8 μg each of appropriate constructs using Lipofectamine 2000 (Thermo Fisher Scientific). Approximately 24 h post-transfection, cells were scraped directly into 200 μl of lysis buffer (50 mM Tris, 300 mM NaCl, 5 mM MgCl₂, 1% NP-40, 7 mM BME), plus protease inhibitors. Twenty microliter of cell lysate was saved for input controls. The remainder of the lysate was added to 5 μl of Pierce Protein G agarose beads (Thermo Fisher Scientific), and the mixture was rotated 1 to 2 h at 4 °C to preclear. Following the preclear incubation, the mixtures were spun quickly, and the super was added to 40 μl of EZ View Red anti-FLAG M2 agarose beads (Millipore Sigma). The mixture was rotated 1 to 2 h at 4 °C and washed 3× with PBS + 0.1% NP-40 + 500 mM NaCl. The immunoprecipitated proteins were eluted from the beads in 5× Laemmli buffer and boiled at 95 °C for 10 min. The bead-free IP supernatant and the input samples were run on 10% SDS-PAGE gels and analyzed with Western blots as described above.