title: β-Actin as a loading control: Less than 2 μg of total protein should be loaded.
abstract: 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.
authors.affiliation: Guangdong Province Key Laboratory for Biotechnology Drug Candidates, School of Biosciences and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, P. R. China.
authors.forename: Wei
authors.initials: W
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, P. R. China.
authors.forename: Wei-Hua
authors.initials: WH
authors.lastname: Xu
mesh_terms: D000199:Actins / Q000032:analysis*; D015153:Blotting, Western / Q000379:methods* / Q000592:standards*; D016014:Linear Models; D012015:Reference Standards; D012680:Sensitivity and Specificity
chemical_list: D000199:Actins
keywords: Linearity range; Loading control; Western blot analysis; β-Actin