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, also 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. resolving the mechanisms underpinning adaptations to exercise). This is none more so than in relation to skeletal muscle physiology (i.e. The applications of Western/immuno-blotting (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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