Journal
PLOS ONE
Volume 8, Issue 8, Pages -Publisher
PUBLIC LIBRARY SCIENCE
DOI: 10.1371/journal.pone.0072457
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Categories
Funding
- Biotechnology and Biological Sciences Research Council (BBSRC) UK through a studentship award (CJO)
- BBSRC
- SMA Trust
- Muscular Dystrophy Campaign
- BBSRC [BBS/E/D/20251967, BBS/E/D/20251969, BBS/E/D/20251968] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council [BBS/E/D/20251968, 1097988, BBS/E/D/20251969, BBS/E/D/20251967] Funding Source: researchfish
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Western blotting has been a key technique for determining the relative expression of proteins within complex biological samples since the first publications in 1979. Recent developments in sensitive fluorescent labels, with truly quantifiable linear ranges and greater limits of detection, have allowed biologists to probe tissue specific pathways and processes with higher resolution than ever before. However, the application of quantitative Western blotting (QWB) to a range of healthy tissues and those from degenerative models has highlighted a problem with significant consequences for quantitative protein analysis: how can researchers conduct comparative expression analyses when many of the commonly used reference proteins (e.g. loading controls) are differentially expressed? Here we demonstrate that common controls, including actin and tubulin, are differentially expressed in tissues from a wide range of animal models of neurodegeneration. We highlight the prevalence of such alterations through examination of published -omics'' data, and demonstrate similar responses in sensitive QWB experiments. For example, QWB analysis of spinal cord from a murine model of Spinal Muscular Atrophy using an Odyssey scanner revealed that beta-actin expression was decreased by 19.3 +/- 2% compared to healthy littermate controls. Thus, normalising QWB data to beta-actin in these circumstances could result in 'skewing' of all data by similar to 20%. We further demonstrate that differential expression of commonly used loading controls was not restricted to the nervous system, but was also detectable across multiple tissues, including bone, fat and internal organs. Moreover, expression of these control'' proteins was not consistent between different portions of the same tissue, highlighting the importance of careful and consistent tissue sampling for QWB experiments. Finally, having illustrated the problem of selecting appropriate single protein loading controls, we demonstrate that normalisation using total protein analysis on samples run in parallel with stains such as Coomassie blue provides a more robust approach.
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