Ni nta affinity resin column

The secreted reporters were stably expressed in isogenic HEK293-6E cell lines selected by two weeks of culture in the presence of 0.32 µg/mL G418 (Sigma-Aldrich) and two rounds of FACS enrichment for GFP expression. A stable pool of cells was seeded at a density of 0.25 × 10⁶ cells/mL and cultured for 5 days on an orbital shaker in F17 medium (Gibco) supplemented with 0.1 Kolliphor P188 (Sigma-Aldrich) and 2% Glutamax. Culture medium containing secreted mucin TR reporter was harvested (3000×g, 10 min), mixed 3:1 (v/v) with 4× binding buffer (100 mM sodium phosphate, pH 7.4, 2 M NaCl), and run through a nickel-nitrilotriacetic acid (Ni-NTA) affinity resin column (Qiagen), pre-equilibrated with washing buffer (25 mM sodium phosphate, pH 7.4, 500 mM NaCl, 20 mM imidazole). The column was washed multiple times with washing buffer and mucin TR reporter was eluted with 200 mM imidazole. Eluted fractions were analyzed by SDS-PAGE and fractions containing the mucin TR reporter were desalted followed by buffer exchange to MiliQ using Zeba spin columns (ThermoFisher Scientific). Yields were quantified using a Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific) following the manufacturer’s instructions and NuPAGE Novex Bis-Tris (4–12%, ThermoFisher Scientific) Coomassie blue analysis.

HEK293-6E cells were seeded at a cell density of 0.5 × 10⁶ cells/ml and transfected with FcγRIIIa-158F/V with 10X his-tag and AviTag™ the next day with 1:3 μg DNA : PEI per 1 × 10⁶ cells. Secreted protein was harvested after 72 hours and purified from culture medium by nickel affinity chromatography. For this, the culture medium was centrifuged, filtered (0.45 μm), mixed 3:1 (v/v) in 4X binding buffer (100 mM sodium phosphate, pH 7.4, 2 M NaCl) and applied to self-packed nickel-nitrilotriacetic acid (Ni-NTA) affinity resin column (Qiagen), which was pre-equilibrated in washing buffer (25 mM sodium phosphate, pH 7.4, 500 mM NaCl, 20 mM imidazole). After washing, bound protein was eluted with 250 mM imidazole in washing buffer. Purity and quantification were evaluated by SDS-PAGE and Coomassie staining. Purified protein was buffer-exchanged to approximately 1 mg/ml in 50mM AmBic buffer with 2 ml Zeba Spin Desalting Column 7K MWCO (ThermoFisher).