Irdye 680 conjugated goat anti mouse secondary antibody

The general procedure for Western blot protein analysis has been described previously (Gregg et al. 2016 (link)). After tissue sample preparation, equal amounts of protein (20 μg per loading sample, as determined using a Thermo Scientific NanoDrop 2000 spectrometer) were loaded onto 7.5% Mini Protean Precast gels (Bio-Rad, Hercules, CA) for separation and transferred onto nitrocellulose membranes (Life Technologies, Carlsbad, CA). Membranes were blocked for 1-h at room temperature in Odyssey Blocking Buffer (Li-Cor Biosciences, Lincoln, NE), followed by overnight incubation at 4°C with the GCPII primary antibody (1:5,000, mouse monoclonal; GeneTex, Irvine, CA). The next day, membranes were washed with Tween-Tris buffered saline (TTBS) 3 times for 8 min, followed by 90 min incubation with β-tubulin loading control primary antibody at room temperature (1:800,000, monoclonal mouse; Cell Signaling, Danvers, MA). Membranes were washed again with TTBS, and then incubated for 1-h at room temperature with IRDye 680-conjugated goat anti-mouse secondary antibody (1:10,000; Li-Cor Biosciences). Membranes were given 3 final TTBS washes, before proteins were detected and quantified using the Odyssey infrared imaging system (Li-Cor Biosciences). The relative density of each sample (GCPII/β-tubulin optical densities) were determined and averaged for each group.