β-tubulin is a more suitable internal control than β-actin in Western blot analysis of spinal cord tissues after traumatic injury

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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 ± 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.

Original languageEnglish (US)
Pages (from-to)1794-1801
Number of pages8
JournalJournal of Neurotrauma
Volume23
Issue number12
DOIs
StatePublished - Dec 2006
Externally publishedYes

Fingerprint

Tubulin
Spinal Cord Injuries
Actins
Spinal Cord
Western Blotting
Wounds and Injuries
Essential Genes
Proteins
Central Nervous System

Keywords

  • β-actin
  • β-tubulin
  • Internal control
  • Spinal cord injury
  • Western blot

ASJC Scopus subject areas

  • Clinical Neurology
  • Neuroscience(all)

Cite this

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title = "β-tubulin is a more suitable internal control than β-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 ± 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.",
keywords = "β-actin, β-tubulin, Internal control, Spinal cord injury, Western blot",
author = "Naikui Liu and Xiao-Ming Xu",
year = "2006",
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doi = "10.1089/neu.2006.23.1794",
language = "English (US)",
volume = "23",
pages = "1794--1801",
journal = "Journal of Neurotrauma",
issn = "0897-7151",
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AU - Liu, Naikui

AU - Xu, Xiao-Ming

PY - 2006/12

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N2 - 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 ± 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.

AB - 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 ± 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.

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