Quantifying Cochlear Protein Expression

Cochlea proteins were incubated in cell lysis buffer (10 mM Tris, pH=7.4, 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 0.2 mM PMSF) and extracted using a homogenizer. The proteins from the samples (50 μg) were subjected to SDS-polyacrylamide gel electrophoresis and blotted onto a polyvinylidene difluoride membrane. The primary antibodies used were anti-p-Akt rabbit monoclonal antibodies (Millipore; 1 : 200), anti-p27^kip rabbit polyclonal antibodies (Abcam; 1 : 200), and anti-GAPDH mouse monoclonal antibodies (Millipore; 1 : 3333). Subsequently, the membrane was incubated for 1 h at room temperature with the appropriate secondary antibodies coupled with horseradish peroxidase. Finally, the immunoreactive bands were visualized using ECL detection reagents.