Abstract
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 β-actin in these circumstances could result in ‘skewing’ of all data by ∼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.
Highlights
Biochemical analysis using Western blotting is an essential tool in determining relative protein expression in complex biological samples
In order to obtain an initial estimate for the variability of expression levels of commonly used loading control proteins in experimental studies, we first undertook an examination of published protein expression data from a range of human and animal model studies including four of our own published datasets (Table 1; [6,10,11,12,13,14,15,16])
We began with a focus on the expression levels of commonly used loading control proteins within the nervous system, as this is our specific area of research interest
Summary
Biochemical analysis using Western blotting is an essential tool in determining relative protein expression in complex biological samples. It is often used in conjunction with mass screening technologies such as proteomics to confirm differential candidate expression in various models of disease. Together with increasingly sophisticated in vivo and in vitro biological models, quantitative protein expression analyses are frequently being employed in attempts to elucidate the molecular mechanisms regulating cellular form and function in health and disease. With the development of more sensitive fluorescent labelling, which demonstrates a greater quantifiable linear range, sensitivity and stability in comparison to conventional ECL detection [2], analysis of protein expression can be justifiably termed ‘‘quantitative’’. The leading company in this market is LICOR and its Odyssey fluorescence imaging scanner appears to have the most significant market penetration at present with 206 instruments currently installed in the UK alone (Personal communication; LICOR technical consultants)
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