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β‐Actin as a loading control: Less than 2 μg of total protein should be loaded
Author(s) -
Chen Wei,
Xu WeiHua
Publication year - 2015
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201500138
Subject(s) - western blot , normalization (sociology) , actin , blot , antibody , microbiology and biotechnology , chemistry , biology , biophysics , biochemistry , immunology , gene , sociology , anthropology
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.