Semi-Quantification of proteins using Western blots typically involves normalization against housekeeping genes such as β-actin. More recently, ponceau S and Coomassie blue staining have both been shown to be suitable alternatives to housekeeping genes as loading controls. Stain free total protein staining offers the advantage of no staining or destaining steps. Evaluation of the use of Stain free staining as an alternative to β-actin or the protein stain ponceau S showed that Stain free staining was superior to β-actin and as good as or better than ponceau S staining as a loading control for Western blots.

Summary Western blotting is one of the most commonly used laboratory techniques for identifying proteins and semi-quantifying protein amounts, however, several recent findings suggest that western blots may not be as reliable as previously assumed. This is not surprising since many labs are unaware of the limitations of western blotting. In this manuscript we review essential strategies for improving confidence in the accuracy of western blots. These strategies include selecting the best normalization standard, proper sample preparation, determining the linear range for antibodies and protein stains relevant to the sample of interest, confirming the quality of the primary antibody, preventing signal saturation and accurately quantifying the signal intensity of the target protein. Although western blotting is a powerful and indispensable scientific technique that can be used to accurately quantify relative protein levels, it is necessary that proper experimental techniques and strategies are employed.

Western blotting is a commonly used technique in biological research. A major problem with Western blotting is not the method itself, but the use of poor quality antibodies as well as the use of different experimental conditions that affect the linearity and sensitivity of the Western blot. Investigation of some conditions that are commonly used and often modified in Western blotting, as well as some commercial antibodies, showed that published articles often fail to report critical parameters needed to reproduce the results. These parameters include the amount of protein loaded, the blocking solution and conditions used, the amount of primary and secondary antibodies used, the antibody incubation solutions, the detection method and the quantification method utilized. In the present study, comparison of ubiquitinated proteins in rat heart and liver samples showed different results depending on the antibody utilized. Validation of five commercial ubiquitin antibodies using purified ubiquitinated proteins, ubiquitin chains and free ubiquitin showed that these antibodies differ in their ability to detect free ubiquitin or ubiquitinated proteins. Investigating proteins modified with interferon-stimulated gene 15 (ISG15) in young and old rat hearts using six commercially available antibodies showed that most antibodies gave different semi-quantitative results, suggesting large variability among antibodies. Evidence showing the importance of the Western blot buffer and the concentration of antibody used is presented. Hence there is a critical need for comprehensive reporting of experimental conditions to improve the accuracy and reproducibility of Western blot analysis. A Western blotting minimal reporting standard (WBMRS) is suggested to improve the reproducibility of Western blot analysis.

The ubiquitin-proteasome system (UPS) is the major intracellular degradation system, and its proper function is critical to the health and function of cardiac cells. Alterations in cardiac proteasomes have been linked to several pathological phenotypes, including cardiomyopathies, ischemia-reperfusion injury, heart failure, and hypertrophy. Defects in proteasome-dependent cellular protein homeostasis can be causal for the initiation and progression of certain cardiovascular diseases. Emerging evidence suggests that the UPS can specifically target proteins that govern pathological signaling pathways for degradation, thus altering downstream effectors and disease outcomes. Alterations in UPS-substrate interactions in disease occur, in part, due to direct modifications of 19S, 11S or 20S proteasome subunits. Post-translational modifications (PTMs) are one facet of this proteasomal regulation, with over 400 known phosphorylation sites, over 500 ubiquitination sites and 83 internal lysine acetylation sites, as well as multiple sites for caspase cleavage, glycosylation (such as O-GlcNAc modification), methylation, nitrosylation, oxidation, and sumoylation. Changes in cardiac proteasome PTMs, which occur in ischemia and cardiomyopathies, are associated with changes in proteasome activity and proteasome assembly; however several features of this regulation remain to be explored. In this review, we focus on how some of the less common PTMs affect proteasome function and alter cellular protein homeostasis.

The ubiquitin-proteasome system (UPS) plays a critical role in removing unwanted intracellular proteins and is involved in protein quality control, signalling and cell death. Because the heart is subject to continuous metabolic and mechanical stress, the proteasome plays a particularly important role in the heart, and proteasome dysfunction has been suggested as a causative factor in cardiac dysfunction. Proteasome impairment has been detected in cardiomyopathies, heart failure, myocardial ischaemia, and hypertrophy. Proteasome inhibition is also sufficient to cause cardiac dysfunction in healthy pigs, and patients using a proteasome inhibitor for cancer therapy have a higher incidence of heart failure. In this Topical Review we discuss the experimental data which suggest UPS dysfunction is a common feature of cardiomyopathies, with an emphasis on hypertrophic cardiomyopathy caused by sarcomeric mutations. We also propose potential mechanisms by which cardiomyopathy-causing mutations may lead to proteasome impairment, such as altered calcium handling and increased oxidative stress due to mitochondrial dysfunction.