Optimising methods for the preservation, capture and identification of ubiquitin chains and ubiquitylated proteins by immunoblotting

Immunoblotting is a powerful technique for the semi-quantitative analysis of ubiquitylation events, and remains the most commonly used method to study this process due to its high specificity, speed, sensitivity and relatively low cost. However, the ubiquitylation of proteins is complex and, when th...

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Bibliographic Details
Published in:Biochemical and biophysical research communications 2015-10, Vol.466 (1), p.1-14
Main Authors: Emmerich, Christoph H., Cohen, Philip
Format: Article
Language:English
Subjects:
Animals
Deubiquitylase
Electrophoresis, Polyacrylamide Gel - methods
Humans
Immunoblotting
Immunoblotting - methods
Immunoprecipitation - methods
Mini Review
topology
Ubiquitin
Ubiquitin - analysis
Ubiquitin - genetics
Ubiquitin - metabolism
Ubiquitin chain topology
Ubiquitin-binding-domain
Ubiquitin-Specific Proteases - metabolism
Ubiquitinated Proteins - analysis
Ubiquitinated Proteins - metabolism
Ubiquitination
Up-Regulation
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Summary:Immunoblotting is a powerful technique for the semi-quantitative analysis of ubiquitylation events, and remains the most commonly used method to study this process due to its high specificity, speed, sensitivity and relatively low cost. However, the ubiquitylation of proteins is complex and, when the analysis is performed in an inappropriate manner, it can lead to the misinterpretation of results and to erroneous conclusions being reached. Here we discuss the advantages and disadvantages of the methods currently in use to analyse ubiquitin chains and protein ubiquitylation, and describe the procedures that we have found to be most useful for optimising the quality and reliability of the data that we have generated. We also highlight commonly encountered problems and the pitfalls inherent in some of these methods. Finally, we introduce a set of recommendations to help researchers obtain high quality data, especially those new to the field of ubiquitin signalling. The specific topics addressed in this article include sample preparation, the separation, detection and identification of particular ubiquitin chains by immunoblotting, and the analysis of ubiquitin chain topology through the combined use of ubiquitin-binding proteins and ubiquitin linkage-specific deubiquitylases.
ISSN:0006-291X
1090-2104
DOI:10.1016/j.bbrc.2015.08.109