Ni nta agarose beads

pET14b expression vector carrying rat 4EBP1 (Addgene #15679; Choi et al., 2003 (link)) was transformed into the BL21 codon(+)RIL E. coli strain. The cells were grown in LB medium at 37°C until the OD₆₀₀ reached 0.4–0.6. Expression of 4EBP1 was then induced by the addition of 0.1 mM isopropyl-β-D-1-thiogalactopyranoside, and the cells were further cultured at 18°C overnight. The cultures were harvested, resuspended in lysis buffer (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 2 mM 2-mercaptoethanol and 10% glycerol) and disrupted by sonication (for 10 min with on-time of 1 s and off-time of 1 s at 4°C). The cell debris was removed by centrifugation for 20 min at 10 000 g, and the supernatant was mixed gently with 1 ml of Ni-NTA agarose beads (Fujifilm Wako) at 4°C for 1 h. The protein-bound beads were packed into an Econo-Column (Bio-Rad Laboratories) and washed with lysis buffer. The proteins were eluted with lysis buffer containing 500 mM imidazole and dialyzed against storage buffer (20 mM Tris-HCl, pH 7.5, 200 mM NaCl, 2 mM 2-mercaptoethanol and 10% glycerol). The samples were flash-frozen in liquid nitrogen and stored at −80°C.