Chen Wei, Xu Wei-Hua
Guangdong Province Key Laboratory for Biotechnology Drug Candidates, School of Biosciences and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, P. R. China.
State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, P. R. China.
Electrophoresis. 2015 Sep;36(17):2046-9. doi: 10.1002/elps.201500138. Epub 2015 Aug 3.
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.
为了在蛋白质印迹分析中进行数据标准化，期刊编辑和审稿人通常要求作者在检测目标蛋白后，用β-肌动蛋白特异性抗体对蛋白质印迹膜进行再检测。然而，在大多数情况下，β-肌动蛋白上样量过多，导致无法检测到蛋白质上样量的差异。在本研究中，我们试图优化用于β-肌动蛋白检测的上样蛋白量，以实现合适的蛋白质印迹分析数据标准化。我们的数据表明，当使用β-肌动蛋白作为上样对照时，上样的总蛋白量应少于2μg。我们还建议避免用β-肌动蛋白特异性抗体对膜进行再检测。
