Hitrap mabselect column

Nimotuzumab-SpyTag and anti-MBP-SpyTag were expressed using the Gibco™ Expi293™ Expression System (Life Technologies, catalog number A14635), according to the manufacturer's protocol. Briefly, 1 day before transfection, Expi293F cells were diluted to 2 × 10 6 cells/ml in Expi293 Expression Medium (Life Technologies). On the day of transfection, 30 μg of plasmid DNA (1:1 ratio) was complexed with 80 μL ExpiFectamine™ 293 reagent. The complexed DNA was then transferred to 7.5 × 10 7 cells (final cell density of 2.5 × 10 6 cells/ml). The next day, enhancers 1 and 2 were added to the media to bring the final volume up to 30 ml. Cells were cultured for 6-7 days. Cells were spun down, and supernatant was collected and filtered through a 0.45-μm-membrane filter (Minisart, Sartorius Stedim). Protein A binding buffer (Sodium Phosphate 20 mM, 0.15 M NaCl, pH 7.2) was added to the supernatant, and the antibody-SpyTag was purified by GE Healthcare AKTA FPLC system using HiTrap MabSelect column (GE healthcare). The antibody-SpyTag was eluted using IgG elution buffer (Fisher Scientific) and neutralized with Neutralization Buffer (1 M Tris-HCl pH 9.0). Antibody-SpyTag was dialyzed overnight with phosphate-buffered saline (PBS) and concentrated using a 30 K MWCO filter (Millipore). Fragments were filter sterilized and stored at -80 °C.