An overview of technical considerations for Western blotting applications to physiological research

Bass, Joseph J.; Wilkinson, Daniel J.; Rankin, Debbie; Phillips, Bethan E.; Szewczyk, Nathaniel J.; Smith, Kenneth; Atherton, Philip J.

Abstract

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. This is none more so than in relation to skeletal muscle physiology (i.e. resolving the mechanisms underpinning adaptations to exercise). Indeed, the inclusion of WB data is now considered an essential aspect of many such physiological publications to provide mechanistic insight into regulatory processes. 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. 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. 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. We aim to provide the reader with improved expertise to critically conduct, evaluate and troubleshoot the WB process, to produce reproducible and reliable blots.

Citation

Bass, J. J., Wilkinson, D. J., Rankin, D., Phillips, B. E., Szewczyk, N. J., Smith, K., & Atherton, P. J. (in press). An overview of technical considerations for Western blotting applications to physiological research. Scandinavian Journal of Medicine and Science in Sports, 27(1), https://doi.org/10.1111/sms.12702

• Journal Article Type: Article
• Acceptance Date: Apr 25, 2016
• Online Publication Date: Jun 5, 2016
• Deposit Date: Jun 13, 2016
• Publicly Available Date: Jun 13, 2016
• Journal: Scandinavian Journal of Medicine & Science in Sports
• Print ISSN: 0905-7188
• Electronic ISSN: 1600-0838
• Publisher: Wiley
• Peer Reviewed: Peer Reviewed
• Volume: 27
• Issue: 1
• DOI: https://doi.org/10.1111/sms.12702
• Keywords: Westen blot, physiology, SDS PAGE, skeletal muscle
• Public URL: https://nottingham-repository.worktribe.com/output/796489
• Publisher URL: http://onlinelibrary.wiley.com/doi/10.1111/sms.12702/abstract
• Contract Date: Jun 13, 2016