β-Tubulin Is a More Suitable Internal Control thanβ-Actin in Western Blot Analysis of Spinal Cord Tissues after Traumatic Injury

Abstract

Western blot is a widely used method for determining specific protein levels. To control and correct for loading error, an internal control is often used. To date, two housekeeping gene–coded proteins (i.e., β-actin and β-tubulin) are widely used as internal controls in the Western blot analysis. However, no information is available concerning the stability of their expressions in response to a traumatic injury to the central nervous system (CNS). If so, their use as an internal control may have a negative impact on data acquisition, analysis, and interpretation. Using Western blot analysis, we demonstrated that spinal cord injury (SCI) induced a significant increase in β-actin expression which peaked at 7 days post-SCI (2.48-fold). Coefficient of variation (CV) analysis showed that the CV of β-actin expression was 43.79 ± 4.67%, significantly higher than that of six loadings from a single sample (6.5 ± 0.9%, p < 0.01), indicating that increased expression of β-actin was a result of SCI, instead of a loading error. In contrast, no statistically significant difference was found in β- tubulin expression following SCI, compared with sham-operated controls. The CV of β-tubulin expression following SCI was 14.3 β 3.96%, significantly less than that of the β-actin expression (43.79 ± 4.67%; p < 0.01). Taken together, our study suggests that β-actin whose expression increases following SCI is not a suitable internal control for Western blot analysis of spinal cord tissues following a traumatic injury. In contrast, β-tubulin, whose expression was not significantly affected by SCI, is a better choice for the internal control.