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Western blotting revisited: Critical perusal of underappreciated technical issues


Gorr, Thomas A; Vogel, Johannes (2015). Western blotting revisited: Critical perusal of underappreciated technical issues. Proteomics. Clinical applications, 9(3-4):396-405.

Abstract

The most commonly used semiquantitative analysis of protein expression still employs protein separation by denaturing SDS-PAGE with subsequent Western blotting and quantification of the resulting ODs of bands visualized with specific antibodies. However, many questions regarding this procedure are usually ignored, although still in need of answering: Does isolation or separation procedure harm the integrity or affect modifications (e.g., phosphorylation) of the protein of interest? Does denaturation reduce binding of antibodies used for detection? Should denaturation be performed or should a native gel be run? How can artificial degradations or aggregations be distinguished from biological relevant ones? If the antibody detects multiple bands (which is not uncommon), which one(s) should be taken into account for quantification and why? Which loading control protein should be chosen and is it really "housekeeping" and how can this be verified? Is the image acquisition system linear and does it come with a sufficient dynamic range? How to account and control for background staining? This article is intended to address these questions and raise the readers awareness to possible Western blot alternatives in the attempt of minimizing possible pitfalls that might loom anywhere from protein isolation to acquisition of final quantitative data.

Abstract

The most commonly used semiquantitative analysis of protein expression still employs protein separation by denaturing SDS-PAGE with subsequent Western blotting and quantification of the resulting ODs of bands visualized with specific antibodies. However, many questions regarding this procedure are usually ignored, although still in need of answering: Does isolation or separation procedure harm the integrity or affect modifications (e.g., phosphorylation) of the protein of interest? Does denaturation reduce binding of antibodies used for detection? Should denaturation be performed or should a native gel be run? How can artificial degradations or aggregations be distinguished from biological relevant ones? If the antibody detects multiple bands (which is not uncommon), which one(s) should be taken into account for quantification and why? Which loading control protein should be chosen and is it really "housekeeping" and how can this be verified? Is the image acquisition system linear and does it come with a sufficient dynamic range? How to account and control for background staining? This article is intended to address these questions and raise the readers awareness to possible Western blot alternatives in the attempt of minimizing possible pitfalls that might loom anywhere from protein isolation to acquisition of final quantitative data.

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Additional indexing

Item Type:Journal Article, refereed, further contribution
Communities & Collections:05 Vetsuisse Faculty > Institute of Veterinary Physiology
Dewey Decimal Classification:570 Life sciences; biology
Language:English
Date:16 January 2015
Deposited On:15 Apr 2015 15:17
Last Modified:08 Dec 2017 12:48
Publisher:Wiley-Blackwell Publishing, Inc.
ISSN:1862-8346
Publisher DOI:https://doi.org/10.1002/prca.201400118
PubMed ID:25597284

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