Hitrap chelating hp column

SOD3 was purified as previous described^{38 (link)}. Briefly, SOD3 expression plasmid was transfected into HEK-293T cells (ATCC® CRL-11268™) with Attractene (Qiagen) based on the manufacturer’s instructions. Five days after transfection, culture media containing SOD3 were collected, filtrated, and loaded onto HiTrap Chelating HP column (GE Healthcare). After loading, the column was washed with more than 50 column volumes of washing buffer, 50 mM NaPO₄, 500 mM NaCl, and 30 mM imidazole. Then, SOD3 was eluted by the elution buffer containing 50 mM NaPO₄, 500 mM NaCl, 500 mM imidazole, followed by dialysis in PBS containing 50 μM Cu²⁺/Zn²⁺ ions. The concentration of purified SOD3 was determined based on a BSA standard curve with a protein assay dye (Bio-Rad). The recombinant SOD3 was verified by western blot with SOD3 antibody as previous described^{43 (link)}.
The SOD3 enzymatic activity was measured by the Superoxide Dismutase Assay Kit-WST (Dojindo Molecular Technologies, MD, USA) following the manufacturer’s instructions. Briefly, a 20 µl sample or PBS (bank control) was mixed with 200 µl of 200 µM WST working solution and 20 µl of enzyme working solution. The mixtures were incubated for 20 min for developing the signal, which was read at A₄₅₀ using a micro-plate reader. The SOD activity was determined from the dilution factor exhibiting 50% inhibition (IC₅₀).

Tag-free DX2 protein was purified as previously described [17 (link)]. C-terminal his-tagged DX2_34-251 and AIMP2_34-320 genes were cloned into pET-28a vector and overexpressed in E. coli BL21(DE3) by induction with 0.5 mM IPTG. After centrifugation, cells were resuspended and sonicated in buffer (35 mM imidazole, 500 mM NaCl, and 20 mM Tris-HCl (pH 7.5)) supplemented with PMSF (phenylmethylsulfonyl fluoride) and protease inhibitor cocktail (Calbiochem, San Diego, CA, USA). After centrifugation, the supernatant was loaded onto a HiTrap chelating HP column (GE Healthcare, Chicago, IL, USA) pre-equilibrated with the same buffer without the protease inhibitors. The column was washed with the equilibration buffer, and the protein was eluted with the elution buffer (1 M imidazole, 500 mM NaCl, and 20 mM Tris-HCl (pH 7.5)). The partially purified proteins were further subjected to size exclusion (HiLoadl 6/600 Superdex75, GE Healthcare, Chicago, IL, USA) and cation exchange (HiTrap Sp HP) chromatographies.

SOD3 was purified as previously described (15 (link)). Briefly, SOD3 plasmids were transfected into human embryonic kidney cells (HEK293E) and media were collected after every 48 h. The collected media were filtered and loaded on HiTrap Chelating HP column (GE Healthcare).The recombinant SOD3 was verified by western blot with SOD3 antibody as previous described (16 (link)). SOD3 activity in the culture medium was analyzed by SOD assay kit as per manufacturer’s instruction (S311, Dojindo Molecular Technologies, Japan).