β-tubulin is a more suitable internal control than β-actin in Western blot analysis of spinal cord tissues after traumatic injury
β-tubulin is a more suitable internal control than β-actin in Western blot analysis of spinal cord tissues after traumatic injury
复制标题
DOI：
10.1089/neu.2006.23.1794
复制
发表时间：
2006-12-01
影响因子：
4.2
通讯作者：
Xu, Xiao-Ming
中科院分区：
文献类型：
--
作者：
Liu, Nai-Kui;Xu, Xiao-Ming
蛋白质印迹是一种广泛使用的测定特定蛋白质水平的方法。为了控制和纠正加载误差，通常使用内部控制。迄今为止，两种看家基因编码的蛋白质（即β-肌动蛋白和β-微管蛋白）被广泛用作蛋白质印迹分析中的内部对照。然而，尚无关于其表达对中枢神经系统（CNS）创伤性损伤的反应稳定性的信息。如果是这样，将它们用作内部控制可能会对数据采集、分析和解释产生负面影响。使用蛋白质印迹分析，我们证明脊髓损伤 (SCI) 诱导 β-肌动蛋白表达显着增加，并在 SCI 后 7 天达到峰值（2.48 倍）。变异系数（CV）分析显示，β-actin表达的CV为43.79 +/- 4.67%，显着高于单个样品的六次上样的CV（6.5 +/- 0.9%，p < 0.01），表明β-actin表达增加是SCI的结果，而不是上样误差。相反，与假手术对照相比，SCI 后 β-微管蛋白表达没有发现统计学上的显着差异。 SCI 后 β-微管蛋白表达的 CV 为 14.3 +/- 3.96%，显着低于 β-肌动蛋白表达的 CV (43.79 ± 4.67%；p < 0.01)。综上所述，我们的研究表明，SCI 后表达增加的 β-肌动蛋白不适合用于创伤性损伤后脊髓组织的蛋白质印迹分析。相比之下，β-微管蛋白的表达不受 SCI 的显着影响，是内参的更好选择。
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., beta-actin and beta-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 beta-actin expression which peaked at 7 days post-SCI (2.48-fold). Coefficient of variation (CV) analysis showed that the CV of beta-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 beta-actin was a result of SCI, instead of a loading error. In contrast, no statistically significant difference was found in beta-tubulin expression following SCI, compared with sham-operated controls. The CV of beta-tubulin expression following SCI was 14.3 +/- 3.96%, significantly less than that of the beta-actin expression (43.79 4.67%; p < 0.01). Taken together, our study suggests that beta-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, beta-tubulin, whose expression was not significantly affected by SCI, is a better choice for the internal control.