Kta start protein purification system

FreeStyle 293-F cells were split to a density of 0.8 x 10⁶ cells/mL at least one hour before transfection. Cells were transfected using FectoPRO Reagent (Polyplus) following manufacturer instructions at a 1:1 DNA to FectoPRO ratio. 90 μg of plasmid DNA was used for transfection (2:1 ratio of heavy and light chain DNA plasmids) for every 200 mL of cell culture. Transfected cells were incubated in a 37°C, 5% CO₂ shaking incubator for 5 to 7 days to allow for the expression and pairing of heavy and light chain gene products. Transfected cell culture supernatants were collected and filtered through 0.22 μM Steritop filters (Millipore Sigma) before loading onto protein A affinity columns using the ÄKTA start protein purification system (Cytiva Life Sciences). Following loading, samples were washed with 1X phosphate-buffered saline (PBS) then eluted with 100 mM glycine, pH 2.2 and immediately neutralized with 1 M Tris, pH 9.0. Elution fractions were concentrated using Amicon 30K Ultra-0.5 mL Centrifugal Filters (Millipore Sigma) and buffer-exchanged into PBS with PD-10 desalting columns (Cytiva). All purified proteins were validated for integrity and purity via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and stored at -80°C until use.