β-Actin is not a reliable loading control in Western blot analysis

beta-Actin is often used as a loading control in Western blot analysis. We analyzed the ability of beta-actin-specific antibodies to recognize differences in protein loading. We found that, at higher total protein loads as required for the detection of low-abundance proteins, beta-actin-specific antibodies failed to distinguish differences in actin protein levels. Diluting the antibody working solution or changing the incubation time had little effect on this phenomenon. This shows that beta-Actin is not a reliable loading control in Western blot analysis. In general, it appeared that, at longer incubation times, antibodies seem to be less able to pick up differences in the level of its target protein.