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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.

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Authors

JOSEPH BASS Joseph.Bass@nottingham.ac.uk
Assistant Professor (Physiology and Endocrinology)

Daniel J. Wilkinson

Debbie Rankin

BETH PHILLIPS beth.phillips@nottingham.ac.uk
Professor of Translational Physiology

Nathaniel J. Szewczyk

KENNETH SMITH KEN.SMITH@NOTTINGHAM.AC.UK
Professor of Metabolic Mass Spectrometry

Philip J. Atherton



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.

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

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