Donkey anti goat igg hrp

Cell lysates prepared using mammalian protein extraction reagent (M-PER®; Pierce Biotechnology, Rockford, IL) were mixed with 2× Laemmli sample buffer (Bio-Rad Laboratories, Hercules, CA), boiled for 5 min and separated on a precast 12% SDS-PAGE gel (Bio-Rad Laboratories, Hercules, CA). Proteins were transferred on PVDF membranes, blocked with 5% w/v skim milk and probed overnight (o/n) with antibodies against β-actin (Abcam, Cambridge, MA) and desmin (Abcam, Cambridge, MA) at 4°C with gentle shaking. Membranes were washed with tris-buffered-saline (TBS) containing 0.1% w/v tween and probed with goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology, Dallas, TX) and donkey anti-goat IgG-HRP (Abcam, Cambridge, MA) for 1 h at room temperature (RT) with gentle shaking before visualizing the protein bands using Pierce™ ECL Western Blotting substrate (Thermo Fisher Scientific, Waltham, MA).