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

Abstract

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. 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.

Keywords: SDS PAGE; Westen blot; physiology; skeletal muscle.

Figure 1

The sequential stages of the western blot process.

Figure 2

Representative blots for multiple housekeeping proteins and the Coomassie stained membrane of IGF-1 treated C2C12 cells, demonstrating potential variability in house keeping proteins in response to a treatment.

Figure 3

Examples of image manipulation techniques upon representative blots. A. Multiple blots spliced together to give the impression of a single blot. B. An over saturated blot with no clear distinction between bands of interest. C. Contrast adjustment masking additional bands. D. Image manipulation to intensify, reduce or remove bands.