Hitrap q fast flow column

Separation of LDL(–), an electronegative LDL subfraction, was performed as previously reported with slight modifications [20 (link),23 (link)]. Native LDL was passed through a desalting column (HiTrap™ Desalting; GE Healthcare, Chicago, IL, USA) using buffer A [10 mM Tris-HCl/1 mM EDTA (pH 7.4)]. Approximately 6 mg native LDL was placed on a HiTrap™ Q Fast Flow column (17-5053-01, GE Healthcare, Chicago, IL, USA) followed by stepwise elution using 0 to 1.0 M NaCl. The peak of absorbance at the wavelength of 280 nm eluted with 0.5 M NaCl was collected as the LDL(–) fraction. The recovered LDL(–) fractions were gently mixed with 0.5 mg/mL KBr to adjust the density to 1.063 and then centrifuged at 500,000× g for 2.5 h. The floating LDL(–) fraction was collected, dialyzed against E-PBS, and stored at 4 °C.

The HLA-B*57:01, HLA-B*57:03, HLA-B*58:01 and β₂m genes were sub-cloned into the pET-30 expression vector and were expressed into inclusion bodies separately in Escherichia coli. The HLA complexes were refolded in the presence of the peptides listed in Table 1 and purified as described previously^{63 (link)}. Briefly, 90 mg HLA heavy chain was refolded by rapid dilution in a solution containing 3 M urea (Sigma-Aldrich, USA), 100 mM Tris-HCl, pH 8.0 (Sigma-Aldrich, USA), 400 mM l-arginine-HCl, 5 mM reduced glutathione (Sigma-Aldrich, USA) and 0.5 mM oxidised glutathione (Sigma-Aldrich, USA) in the presence of 30 mg β₂m and 10 mg of the appropriate peptide for 48 h. The refolded HLA-peptide complexes were dialysed into 10 mM Tris, pH 8.0, and purified by size-exclusion chromatography using HiLoad 16/60 Superdex 200 pg (GE Healthcare, USA) columns on an AKTA Purifier (GE Healthcare, USA) FPLC chromatography systems in 10 mM Tris, pH 8.0, and 150 mM NaCl buffer. Final purification was by anion exchange using a HiTrap Q Fast Flow column (GE Healthcare, USA) on the same AKTA system in 10 mM Tris pH 8.0 buffer with a NaCl gradient from 0 to 500 mM over 45 min.