Peroxidase conjugated goat anti rabbit antibody

Cells were collected with phosphate-buffered saline (PBS)-EDTA and resuspended in 70 to 100 μL of lysis buffer (10 mM Tris/HCl, pH 7.5, 150 mM NaCl, 0.5% triton X-100, 0.5% deoxycholate, 5 mM EDTA). Protein concentrations were determined by the Bradford method [59 (link)] and 20–40 μg proteins were loaded onto 10% of SDS-PAGE gels which were run at 100 volts for 2–2.5 h. Proteins were then transferred onto nitrocellulose membranes for 60–120 min at 90 V). Protein transfer was verified by Ponceau red staining, and nitrocellulose membranes were subsequently incubated in 5% non-fat milk blocking solution for 45 min. Membranes were then incubated with primary antibodies directed against βAPP (dilution 1/2000), ADAM10 (dilution 1/500), BACE1 (dilution 1/1000), or β-actin (dilution 1/5000) on a platform shaker overnight at 4 °C. Bound antibodies were detected using goat anti-mouse (dilution 1/3000, polyclonal 7076, Cell Signaling) or goat anti-rabbit peroxidase-conjugated antibody (dilution 1/3000, polyclonal 7074, Cell Signaling). After 3 washes with PBST, membranes were incubated with a HRP-conjugated anti-rabbit (ADAM10, βAPP and BACE1) or anti-mouse (β-actin) secondary antibody (1/3000) for 2 h, rinsed 3 times with PBST, and processed as described above. All protein levels were normalized using β-actin as an internal standard.