The method of sample collection depends upon the sample type skeletal muscle tissue by biopsy (i.e. Whilst performing a western blot, there are multiple key aspects to each step: In the main we will concentrate on analysis of skeletal muscle tissues derived from skeletal muscle biopsies nonetheless, the details described in each element are inherently applicable to other tissues or sample types.
In this review we will describe the stages of the WB, focusing on the more routine WB gel electrophoresis methodologies using standard SDS-PAGE, wet transfers and chemiluminescence, critiquing and highlighting important points to consider throughout. To allow for the greatest accuracy and interpretation of data, each aspect of the WB process must be understood and carefully considered. Originally the process of ‘western blotting’ was the aspect of transferring proteins from a gel to a more stable membrane, although it commonly now refers to the whole process. chemiluminescent or fluorescent), vii) development and detection of the signal, which is theoretically proportional to the degree of antigen/antibody binding and, viii) quantification of the resulting bands using densitometry software (see Figure 1). Outwardly, the principle of the WB is based around a few broad steps: i) the extraction of cellular proteins from a complex mixture of intracellular and extracellular proteins (from tissue, cells etc.), ii) quantification of protein concentration and electrophoretic separation of proteins within a gel matrix, iii) transfer to a membrane with a high affinity for proteins, iv) “blocking” the membrane to reduce non-specific binding, v) antigen detection by antibodies specific for the protein(s) of interest, vi) incubation with a secondary antibody linked to a label (e.g. 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, ageing 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. 2013) and SUMOylation ( Park-Sarge 2010) to name the main applications). 2012), glycosylation ( Péré-Brissaud et al. For example, the WB can be used to investigate protein abundance, kinase activity, cellular localization, protein-protein interactions, or monitoring of post-translational modifications (i.e., events of cleavage, phosphorylation ( Nairn et al. 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.