Total Protein Analysis as a Reliable Loading Control for Quantitative Fluorescent Western Blotting

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.

Transparency Is the Key to Quality

“…This is further complicated by the fact that some detection methods, in particular enhanced chemiluminescence using x-ray film, have a very restricted linear range, and careful attention to the experimental conditions is necessary to ensure linearity. It is typically better to normalize Western blots using total protein loading as the denominator (3)(4)(5)(6)(7)(8)(9)(10)(11). To avoid potential pitfalls and with a focus on improving transparency, the JBC strongly recommends that authors describe their methods used to quantify signal intensity, how the linearity of signal intensity with antigen loading was established, and how protein loading was normalized between lanes.…”

Evaluation of the role of an antioxidant gene in NSC-34 motor neuron-like cells as a model of a motor neuron disease

“…3B). Moreover, a previous study reported no signs of toxicity or any apparent differences in cellular growth rate, even after transduction with higher MOIs (data not provided) [13].…”

“…Ideally, SMA pathophysiology should be studied in in vitro cultures of primary MNs [10,15,18,23]. However, due to decreased MN culture yields, the NSC-34 cell line is considered a suitable alternative to MN cultures and has been widely used to model MN diseases [13]. Indeed, NSC-34 MN-like cells differentiate via a process resembling that of primary MNs, comprise extended neurites, and express MN-specific markers [4].…”

The Pseudomonas aeruginosa Two-Component Regulator AlgR Directly Activates rsmA Expression in a Phosphorylation-Independent Manner

“…The membranes were blocked and probed using a 1:2,500 dilution of anti-HA monoclonal antibody in blocking buffer (Thermo Fisher, Pittsburgh, PA), followed by a 1:30,000 dilution of horseradish peroxidase-conjugated goat anti-rabbit antibody (69). Densitometry of Coomassie-stained SDS-PAGE gels was used to standardize the Western blots using total protein analysis, as described previously (70,71). Detection was performed using the ECL Plus kit (Thermo Fisher) and chemiluminescence detection (ProteinSimple, Santa Clara, CA).…”