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Western blot analysis4/19/2023 ![]() Crucial quality control elements of a WB may be overlooked, leading to poor quality blots, and the potential for unintentionally misleading data production and interpretation. This expansion in WB applications has led to an increased number of users lacking analytical biochemistry backgrounds to appreciate important caveats. This is for reasons relating to the pursuit of an improved understanding of molecular pathways involved in the regulation of transcription and translation by exercise and nutrition in health, aging, and disease. While such WB approaches are routinely used in many fields of biochemical research, the application of the WB to skeletal muscle and exercise physiology is increasing. For example, the WB can be used to investigate protein abundance, kinase activity, cellular localization, protein–protein interactions, or monitoring of post-translational modifications. The Western blot (WB) has diverse applications for investigating regulatory molecular events underpinning energy metabolism, protein turnover and chronic physiological adaptations. We aim to provide the reader with improved expertise to critically conduct, evaluate, and troubleshoot the WB process, to produce reproducible and reliable blots. The present review aims to provide a detailed description and critique of WB procedures and technicalities, from sample collection through preparation, blotting and detection, to analysis of the data collected. This may be due to poor laboratory technique and/or lack of comprehension of the critical steps involved in WB and what quality control procedures should be in place to ensure robust data generation. Despite this popularity, and due to the ubiquitous and relatively inexpensive availability of WB equipment, the quality of WB in publications and subsequent analysis and interpretation of the data can be variable, perhaps resulting in spurious conclusions. Indeed, the inclusion of WB data is now considered an essential aspect of many such physiological publications to provide mechanistic insight into regulatory processes. This is none more so than in relation to skeletal muscle physiology (i.e., resolving the mechanisms underpinning adaptations to exercise). The applications of Western/immunoblotting (WB) techniques have reached multiple layers of the scientific community and are now considered routine procedures in the field of physiology. ![]()
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