Horseradish peroxidase hrp conjugated anti mouse or anti rabbit igg antibodies

MDCK and A549 cells grown on six-well plates, and HBE cells on transwells were lysed in lysis buffer (1% Triton X-100, 10% glycerol, 25 mM HEPES) on ice. A 50-μg sample of protein lysate, as determined by Bradford assay (Thermo-Fisher Scientific), was separated by SDS–4 to 12% morpholineethanesulfonic acid PAGE (Invitrogen). The proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad) and blocked in 5% milk in PBS with 0.1% Tween 20. The membranes were incubated with the primary antibodies (mouse anti-acetylated α-tubulin monoclonal antibody [purchased from Sigma] at 1:1,000, rabbit anti-α-tubulin monoclonal antibody [from Cell Signaling] at 1:1,000) in 3% BSA and 0.04% sodium azide in PBS overnight at 4°C. The membranes were subsequently probed with horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit IgG antibodies (Bio-Rad) in 5% milk and visualized with enhanced chemiluminescence reagents (Bio-Rad).