Standard loading controls are not reliable for Western blot quantification across brain development or in pathological conditions

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.