A Defined Methodology for Reliable Quantification of Western Blot Data

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Abstract

Chemiluminescent western blotting has been in common practice for over three decades, but its use as a quantitative method for measuring the relative expression of the target proteins is still debatable. This is mainly due to the various steps, techniques, reagents, and detection methods that are used to obtain the associated data. In order to have confidence in densitometric data from western blots, researchers should be able to demonstrate statistically significant fold differences in protein expression. This entails a necessary evolution of the procedures, controls, and the analysis methods. We describe a methodology to obtain reliable quantitative data from chemiluminescent western blots using standardization procedures coupled with the updated reagents and detection methods.

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Coomassie staining as loading control in Western blot analysis.

Lars Ekblad, Charlotte Welinder (2011)

In Western blotting, immunodetection of housekeeping proteins is routinely performed to detect differences in electrophoresis loading. The present work describes a much faster and simpler protein staining method, which is compatible with ordinary blocking conditions. In addition, the method can be used after immunodetection with superior linearity compared to ordinary staining methods. After immunoblotting and staining, protein bands can be further identified using peptide mass fingerprinting.

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The use of total protein stains as loading controls: an alternative to high-abundance single-protein controls in semi-quantitative immunoblotting.

Georgina M. Aldridge, David M. Podrebarac, William Greenough … (2008)

Western blots are used to estimate the relative concentrations of proteins of interest based on staining by specific antibodies. Quantitative measurements are often subject to error due to overloading of the loading control and over-reliance on normalization. We have found that at the protein concentrations normally used to quantify most low-abundance proteins of interest, frequently used single-protein loading controls, such as glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and beta-actin, do not accurately reflect differences in protein concentration. Two total protein stains, SYPRO Ruby and Amido Black, were compared and found to be acceptable alternatives to single-protein controls. Although we cannot prove that high-abundance loading controls are inaccurate under all possible conditions, we conclude that the burden of proof should lie with the researcher to demonstrate that their loading control is reflective of quantitative differences in protein concentration.

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Stain-Free technology as a normalization tool in Western blot analysis.

Sabine Hornhardt, Anne Gürtler, Kristin K. McDonald … (2013)

Western blots are used to specifically measure the relative quantities of proteins of interest in complex biological samples. Quantitative measurements can be subject to error due to process inconsistencies such as uneven protein transfer to the membrane. These non-sample-related variations need to be compensated for by an approach known as normalization. Two approaches to data normalization are commonly employed: housekeeping protein (HKP) normalization and total protein normalization (TPN). In this study, we evaluated the performance of Stain-Free technology as a novel TPN tool for Western blotting experiments in comparison with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a representative of the HKP normalization strategy. The target protein (TP) used for this study was MCM7, a DNA licensing replication factor, which was shown previously to be down-regulated by 20% in irradiated lymphoblastoid cell lines (LCLs). We studied the regulation of MCM7 with a multiplex Western blotting approach based on fluorescently labeled secondary antibodies and found that Stain-Free technology appears to be more reliable, more robust, and more sensitive to small effects of protein regulation when compared with HKP normalization with GAPDH. Stain-Free technology offers the additional advantages of providing checkpoints throughout the Western blotting process by allowing rapid visualization of gel separation and protein transfer. Copyright © 2012 Elsevier Inc. All rights reserved.

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Author and article information

Contributors

: sean_taylor@bio-rad.com

Journal

Journal ID (nlm-ta): Mol Biotechnol

Journal ID (iso-abbrev): Mol. Biotechnol

Title: Molecular Biotechnology

Publisher: Springer US (Boston )

ISSN (Print): 1073-6085

ISSN (Electronic): 1559-0305

Publication date (Electronic): 25 May 2013

Publication date PMC-release: 25 May 2013

Publication date (Print): 2013

Volume: 55

Pages: 217-226

Affiliations

[ ]Bio-Rad Laboratories (Canada) Ltd, 1329 Meyerside Dr, Mississauga, ON L5T 1C9 Canada

[ ]Bio-Rad Laboratories, 6000 Alfred Nobel Drive, Hercules, CA 94547 USA

[ ]Bio-Rad Laboratories, 2000 Alfred Nobel Drive, Hercules, CA 94547 USA

Article

Publisher ID: 9672

DOI: 10.1007/s12033-013-9672-6

PMC ID: 3840294

PubMed ID: 23709336

SO-VID: 5e88480f-9ccd-44b8-a0bb-253ca5498266

License:

Open AccessThis article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.