Mono q gl column

SCA2_CAG72 RNA was synthesized using the MEGAscript kit (Ambion) as previously described (Tsoi et al. 2012 (link)) and was PAGE-purified. BIND was subcloned into the pGEX4T2 vector. Both GST-BIND and free GST expression plasmids were expressed in BL21 (DE3) strain Escherichia coli cells. Large scale culture was grown in LB broth with 200 µg/mL ampicillin at 37°C. Protein expression was induced with 0.2 mM IPTG with shaking overnight at 16°C. Pelleted cells were lysed by sonication in 25 mM Tris 8.0, 150 mM NaCl, and 2 mM DTT, and 1 mM PMSF. The soluble fraction was loaded onto a Glutathione Sepharose 4B column (GE Healthcare) and washed with 90 mL lysis buffer. The protein was eluted with buffer containing 10 mM glutathione. The eluate was further purified by Mono-Q GL column (GE Healthcare) and the peak eluted fractions were combined, concentrated, separated in aliquots and flash frozen.

Wild-type and mutant proteins of human NCL–RRM2/3 were purified by similar protocols. Optimal protein expression condition was obtained from E. coli strain BL21 (DE3). Cells were harvested and lysed by sonication in buffer containing 25 mM Tris–HCl (pH 8.0), 300 mM NaCl, 20 mM imidazole, 5% glycerol, 1 mM benzamidine and 1 mM phenylmethylsulfonyl fluoride (PMSF). The supernatant was subjected to Ni²⁺-NTA column and subsequent eluate was further polished by gel filtration chromatography (Superdex-75 16/60 GL, GE Healthcare) and monoQ GL column (GE Healthcare). The final purified protein was kept in buffer containing 20 mM HEPES (pH 7.4) and 100 mM KCl. All the buffers used were prepared in DEPC-treated water.