title: Standard loading controls are not reliable for Western blot quantification across brain development or in pathological conditions.
abstract: A frequently utilized method of data quantification in Western blot analysis is comparison of the protein of interest with a house keeping gene or control protein. Commonly used proteins include β-actin, glyceraldehyde 3 phosphate dehydrogenase (GAPDH), and α-tubulin. Various reliability issues have been raised when using this technique for data analysis-particularly when investigating protein expression changes during development and in disease states. In this study, we have demonstrated that β-actin, GAPDH, and α-tubulin are not appropriate controls in the study of development and hypoxic-ischemic induced damage in the piglet brain. We have also shown that using an in-house pooled standard, loaded on all blots is a reliable method for controlling interassay variability and data normalization in protein expression analysis.
authors.affiliation: Centre for Clinical Research, University of Queensland, Herston, QLD, Australia.
authors.forename: Kate
authors.initials: K
authors.lastname: Goasdoue
authors.affiliation: Centre for Clinical Research, University of Queensland, Herston, QLD, Australia.
authors.forename: Doreen
authors.initials: D
authors.lastname: Awabdy
authors.affiliation: Centre for Clinical Research, University of Queensland, Herston, QLD, Australia.
authors.forename: Stella Tracey
authors.initials: ST
authors.lastname: Bjorkman
authors.affiliation: Centre for Clinical Research, University of Queensland, Herston, QLD, Australia.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Miller
mesh_terms: D000199:Actins / Q000032:analysis; D000818:Animals; D015415:Biomarkers / Q000032:analysis*; D015153:Blotting, Western / Q000592:standards*; D001921:Brain / Q000254:growth & development* / Q000378:metabolism*; D001923:Brain Chemistry; D001927:Brain Diseases / Q000378:metabolism*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000032:analysis; D015203:Reproducibility of Results; D013552:Swine; D014404:Tubulin / Q000032:analysis
chemical_list: D000199:Actins; D015415:Biomarkers; D014404:Tubulin; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases
keywords: Loading control; Pig brain; Standard curve; Western blot