Akta design hplc system

The culture medium was centrifuged at 5000×g for 10 min to obtain the culture supernatant, which was then filtered through a 0.2-μm membrane. The culture supernatants were separated with a molecular weight cut-off (MWCO) spin column (GE Healthcare). The culture supernatant was separated using an AKTA Design HPLC system (GE Healthcare) with a Superdex peptide column (GE Healthcare) and eluted with distilled water at a flow rate of 1 mL/min. The fraction was applied to a Wakopak® Wakosil® C18 and C8 column (Wako Pure Chemical, Osaka, Japan) and eluted with 0.1% formic acid and 0.1% formic acid/acetonitrile in a linear gradient at a flow rate of 2.5 mL/min. The eluent was monitored by ultraviolet spectrophotometry at 210 nm.

The culture medium was centrifuged at 5,000g for 10 min to obtain the culture supernatant, which was then filtered through a 0.2-μm membrane. The culture supernatants were separated with a molecular weight cutoff spin column (GE Healthcare). The >0.5 kDa fraction was obtained by dialysis with a Micro Float-A-Lyzer Dialysis Device (Spectrum Laboratories). The culture supernatant was separated using an AKTA Design HPLC system (GE Healthcare) using a Superdex peptide column (GE Healthcare) and eluted with distilled water at a flow rate of 1 ml min⁻¹. The fraction was applied to an L-column (Chemicals Evaluation and Research Institute, Japan) and eluted with 0.1% formic acid and 0.1% formic acid/acetonitrile in a linear gradient at a flow rate of 0.2 ml min⁻¹. Ion exchange chromatography was performed using a HiTrap-DEAE (GE Healthcare), CM (GE Healthcare) and an SP column (GE Healthcare). The elution buffer for DEAE was 0 M and 1.0 M NaCl in 20 mM Tris-HCl (pH 8.0). The elution buffer for SP and CM was 0 M and 1.0 M NaCl in 50 mM acetic acid in a linear gradient at a flow rate of 1 ml min⁻¹. Finally, the sample was separated using a ZIC-HILIC column (Merck Millipore, Germany) and eluted with 70% acetonitrile and 10% acetonitrile in a linear gradient at a flow rate of 0.5 ml min⁻¹. The eluent was monitored by ultraviolet spectrophotometry at 210 nm.