Blotto milk

Five milliliters of cell media were collected for each sample and concentrated with an Amicon® Ultra-15 Centrifugal Filter Unit. Concentrated cell media was then mixed with Laemmli loading buffer and BME before loading into a pre-cast polyacrylamide SDS-PAGE gel (Bio Rad) and ran at 75 V for 140 min. Western blotting was performed using the Transblot Turbo apparatus and nitrocellulose membrane kit (Bio Rad). Transfer was performed at 25 V for 7 min. Membranes were blocked with 5% Blotto milk (Santa Cruz Biotechnology) in 0.05% TBS-Tween for 1 h at room temperature. Primary staining was performed using a 1:200 dilution of the mouse anti-His sc-8036 antibody (Santa Cruz Biotech) overnight at 4 °C. Blots were then washed three times for 15 min at 4 °C with 0.05% TBS-Tween and stained for 4 h with a 1:1000 dilution of the mouse IgG kappa binding protein (m-IgGκ BP) conjugated to Horseradish Peroxidase (HRP) (Santa Cruz Biotech, sc-516102) at room temperature. After three 15-minute washes, HRP visualization was performed using Super signal west Pico PLUS reagent (Thermo Fisher Scientific). Imaging was performed in a Bio-Rad ChemiDoc MP gel imager. A subsequent epi white light image of the blot under the same magnification was acquired to visualize the stained molecular weight standards.