title: Glyceraldehyde-3-phosphate dehydrogenase is a reliable internal control in Western blot analysis of leukocyte subpopulations from children.
abstract: 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 (β-actin, β-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 β-actin and β-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 β-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 β-actin messenger RNA (mRNA). Further experiments showed that posttranslational regulation was responsible for the low β-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.
authors.affiliation: Department of Pediatrics, Chang Gung Memorial Hospital-Kaohsiung Medical Center, Graduate Institute of Clinical Medical Science, Chang Gung University College of Medicine, Kaohsiung 833, Taiwan.
authors.forename: Hong-Ren
authors.initials: HR
authors.lastname: Yu
authors.forename: Ho-Chang
authors.initials: HC
authors.lastname: Kuo
authors.forename: Hsin-Chun
authors.initials: HC
authors.lastname: Huang
authors.forename: Li-Tung
authors.initials: LT
authors.lastname: Huang
authors.forename: You-Lin
authors.initials: YL
authors.lastname: Tain
authors.forename: Chih-Cheng
authors.initials: CC
authors.lastname: Chen
authors.forename: Chi-Di
authors.initials: CD
authors.lastname: Liang
authors.forename: Jiunn-Ming
authors.initials: JM
authors.lastname: Sheen
authors.forename: I-Chun
authors.initials: IC
authors.lastname: Lin
authors.forename: Chi-Chiang
authors.initials: CC
authors.lastname: Wu
authors.forename: Chia-Yu
authors.initials: CY
authors.lastname: Ou
authors.forename: Kuender D
authors.initials: KD
authors.lastname: Yang
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism; D000328:Adult; D015153:Blotting, Western / Q000379:methods*; D002648:Child; D005312:Fetal Blood / Q000166:cytology*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism*; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D007962:Leukocytes / Q000145:classification / Q000201:enzymology* / Q000276:immunology; D018950:Lipopolysaccharide Receptors / Q000378:metabolism; D014404:Tubulin / Q000235:genetics / Q000378:metabolism
chemical_list: D000199:Actins; D018950:Lipopolysaccharide Receptors; D014404:Tubulin; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases