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BioMed Research International
Volume 2014 (2014), Article ID 361590, 8 pages
http://dx.doi.org/10.1155/2014/361590
Review Article

The Design of a Quantitative Western Blot Experiment

1Bio-Rad Laboratories, 1329 Meyerside Drive, Mississauga, ON, Canada L5T 1C9
2Bio-Rad Laboratories GmbH, Heidemann Street 164, 80939 Munich, Germany

Received 18 December 2013; Accepted 2 February 2014; Published 16 March 2014

Academic Editor: Pavel Hozak

Copyright © 2014 Sean C. Taylor and Anton Posch. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Western blotting is a technique that has been in practice for more than three decades that began as a means of detecting a protein target in a complex sample. Although there have been significant advances in both the imaging and reagent technologies to improve sensitivity, dynamic range of detection, and the applicability of multiplexed target detection, the basic technique has remained essentially unchanged. In the past, western blotting was used simply to detect a specific target protein in a complex mixture, but now journal editors and reviewers are requesting the quantitative interpretation of western blot data in terms of fold changes in protein expression between samples. The calculations are based on the differential densitometry of the associated chemiluminescent and/or fluorescent signals from the blots and this now requires a fundamental shift in the experimental methodology, acquisition, and interpretation of the data. We have recently published an updated approach to produce quantitative densitometric data from western blots (Taylor et al., 2013) and here we summarize the complete western blot workflow with a focus on sample preparation and data analysis for quantitative western blotting.