Purification of Recombinant IgG1 and FcγIIA

The supernatant of the transfected cell cultures were collected a fortnight later and spun down at 4,000 rpm, 4°C, for 1 h before 0.2 µm filtration and purified using the ÄKTA pure system (GE Healthcare) using Protein G affinity column for the IgGs as previously described (15 (link)). The 5 ml HiTrap TALON crude affinity column (GE Healthcare) was used for the recombinant FcγIIA using the same settings. The affinity purified proteins were subsequently gel filtrated using the Superdex 200 pg 16/60 column (GE Healthcare) precalibrated with Gel filtration HMW calibration kit (GE Healthcare) and the Gel filtration LMW calibration kit (GE Healthcare) to extract the monomeric fractions as previously described (14 (link), 15 (link)). Pure IgG1 variants and FcγIIA fractions were collected and concentrated using 100 kDa Amicon Pro System (Merck) and 10 kDa Amicon Pro System (Merck) concentrators, respectively. The final concentrations of the proteins were determined by spectrophotometric analyses using nanodrop (Thermo Fisher Scientific) with consideration of calculated protein extinction coefficients of the various IgG1 variants and FcγIIA.