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

J J Bass,K Smith,P J Atherton,N J Szewczyk,D Rankin,B E Phillips,D J Wilkinson

doi:10.1111/sms.12702

J J Bass, K Smith + Show 5 more

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https://doi.org/10.1111/sms.12702

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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 thispopularity, 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.

Highlights

We will concentrate on analysis of skeletal muscle tissues derived from skeletal muscle biopsies; the details described in each element are inherently applicable to other tissues or sample types
Excessive transfer time will generate a poor signal as proteins pass through both gel and membrane; polyvinylidene difluoride (PVDF) membranes generally have smaller pores compared with nitrocellulose, reducing the amount of protein passing through
Work by Michalewski et al reported that the binding of antiphosphoamino acid antibodies to proteins and membranes critically depended on blocking conditions, with a combination of amicase (5%), bovine serum albumin (BSA) (5%), and membrane-blocking agent (5%) to be most effective in reducing non-specific binding (Michalewski et al, 1999)

Summary

Introduction

The process of “Western blotting” was the aspect of transferring proteins from a gel to a more stable membrane, it commonly refers to the whole process. To allow for the greatest accuracy and interpretation of data, each aspect of the WB process must be understood and carefully considered. We will describe the stages of the WB, focusing on the more routine WB gel electrophoresis methodologies using standard SDSPAGE, wet transfers, and chemiluminescence, critiquing and highlighting important points to consider throughout. Is fluorescent multiplexing suitable? Have the antibodies been stored correctly? Can the membrane be successfully stripped and reprobed?

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