- Open Access
Quantifying western blots: none more black
© The Author(s). 2016
Published: 28 December 2016
Western blotting is among the most common techniques used in molecular biology and a simple way of assessing the presence or absence of a protein. It is also commonly used to compare protein levels in different conditions or in different tissues. This article illustrates some of the easy ways to arrive at a false conclusion when trying to quantify protein levels from western blots.
However, although the two tubulin controls look the same—and give the same intensity measurements using a simple image analysis tool—they do not represent the same underlying expression. In fact, the gel for the wild type was accidentally loaded with more of the sample. The chemiluminescent film was saturated, so the higher level of tubulin in the wild type was not reflected when the intensity measurements were taken: actually when the same amounts of sample were loaded, there was no change in expression of Protein X in the two conditions.
Various methods have been proposed to overcome some of these problems, including using automated quantification platforms, using total protein stains as an alternative control, and optimising steps of the protocol, including protein loading, to ensure a linear dynamic range prior to detection [2, 3]. Complications arising from a lack of linearity in measuring techniques are not only found in western blot experiments of course, and are important to consider in other experimental systems—as the next “What is wrong with this picture?” article will explore.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- de Kok JB, Roelofs RW, Giesendorf BA, Pennings JL, Waas ET, Feuth T, Swinkels DW, Span PN. Normalization of gene expression measurements in tumor tissues: comparison of 13 endogenous control genes. Lab Invest. 2005;85:154–9.View ArticlePubMedGoogle Scholar
- Gilda JE, Gomes AV. Western blotting using in-gel protein labeling as a normalization control: stain-free technology. Methods Mol Biol. 2015;1295:381–91.View ArticlePubMedGoogle Scholar
- Taylor SC, Berkelman T, Yadav G, Hammond M. A defined methodology for reliable quantification of Western blot data. Mol Biotechnol. 2013;55(3):217–26.View ArticlePubMedPubMed CentralGoogle Scholar