Mouse β actin antibody

Cells cultured in 6-well trays were scraped into ice-cold phosphate buffered saline (PBS) and pelleted by centrifugation at 800 x g. After three washes in ice-cold PBS, the cells were lysed for 20 minutes on ice in buffer containing 62.5 mM Tris-HCl (pH 7.4), 1% Triton X-100, 0.1% SDS, 1 mM EDTA, and cOmplete^™ protease inhibitor cocktail (Roche). Samples were then centrifuged for 10 minutes at 16,000 x g and 40 μg of protein supernatant was fractionated by 4–20 % SDS-PAGE using a mini–PROTEAN 3 gel unit (Bio-rad) and then transferred to nitrocellulose membranes. Proteins were detected by probing with the indicated primary antibodies for 2 hours at room temperature, followed by the appropriate secondary antibody conjugated to horseradish peroxidase. Blots were developed using the SuperSignal West Pico Substrate according to the manufacturer’s instructions (Pierce). Primary antibodies used were rabbit anti-ATP7A ^{19 (link)}, rabbit anti-HA (Sigma), mouse β-actin antibody (Abcam), mouse anti-tyrosinase antibody (Santa Cruz Biotechnology). Molecular weight standards were SigmaMarker (Sigma) or PageRuler (ThermoFisher).