β-Actin as a loading control: Less than 2 μg of total protein should be loaded

For the purpose of data normalization in Western blot analysis, journal editors and reviewers usually require authors to reprobe the Western blot membrane with a β-actin-specific antibody after detecting the target protein. In most cases, however, β-actin is overloaded, which results in a failure to detect differences in protein loading. In this study, we attempted to optimize the amount of protein loaded for β-actin detection to permit suitable Western blot analysis data normalization. Our data suggest that less than 2 μg of total protein should be loaded when β-actin is used as a loading control. We also suggest avoiding reprobing the membrane with a β-actin-specific antibody.