Glyceraldehyde-3-phosphate dehydrogenase is a reliable internal control in Western blot analysis of leukocyte subpopulations from children

作者：Yu Hong Ren; Kuo Ho Chang; Huang Hsin Chun; Huang Li Tung; Tain You Lin; Chen Chih Cheng; Liang Chi Di; Sheen Jiunn Ming; Lin I Chun; Wu Chi Chiang; Ou Chia Yu; Yang Kuender D^*

来源：Analytical Biochemistry, 2011, 413(1): 24-29.

DOI：10.1016/j.ab.2011.01.037

• beta-Actin
• GAPDH
• Internal control
• Monocyte
• Neonate
• Western blot

To study differences in the development of immunity, leukocytes from cord blood are often compared with those from adult peripheral blood. Western blot analysis is a common method for detecting proteins. In this study, we investigated the reliability of using different housekeeping proteins (beta-actin, beta-tubulin, and glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) as internal controls for different leukocyte subpopulations from infants, children, and adults. Our results showed that the expression levels of beta-actin and beta-tubulin were much lower in cord blood leukocytes than in adult leukocytes, and this expression pattern persisted in children up to 3 years old. Further study revealed that the beta-actin expression level in newborns was especially lower in CD14-positive monocytes. However, cord blood and adult peripheral blood monocytes had similar expression levels of beta-actin messenger RNA (mRNA). Further experiments showed that posttranslational regulation was responsible for the low beta-actin expression level in neonatal monocytes. Thus, researchers should carefully assess the appropriate use of housekeeping gene-encoded proteins as internal standards to normalize samples for comparisons of different leukocyte populations from subjects of different ages. In this study, we determined that GAPDH was a more reliable internal control than others in Western blot analysis for comparing the development of immunity among infants, children, and adults.

• 出版日期2011-6-1
• 单位长春大学