Each stably-integrated cell line was plated on two 150 × 25 mm tissue culture dishes plates and grown until 70% confluency before cells of each line were combined and harvested for protein extraction via the aforementioned hypotonic lysis protocol. Proteins were again extracted on MagStrep “type 3” XT beads and washed in the same buffer as mentioned above. To ensure all protein was efficiently eluted off of the magnetic resin, the beads were left in 10 mM D-biotin (Buffer BX, IBA LifeSciences) overnight at 4°C. Two one-hour elutions were completed the following day and all elutions were pooled together for each individual sample. The total eluate was then placed on a Spin-X UF 500 μl Centrifugal Concentrator (Corning) and spun at 15,000 × g for approximately 1 hour and 15 min at 4 °C.
