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Biochem Biophys Res Commun. 2015 Oct 9;466(1):1-14. doi: 10.1016/j.bbrc.2015.08.109. Epub 2015 Aug 29.

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

Author information

1
Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, Sir James Black Centre, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom. Electronic address: c.emmerich@dundee.ac.uk.
2
Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, Sir James Black Centre, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.

Abstract

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.

KEYWORDS:

Deubiquitylase; Immunoblotting; Ubiquitin; Ubiquitin chain topology; Ubiquitin-binding-domain

PMID:
26325464
PMCID:
PMC4709362
DOI:
10.1016/j.bbrc.2015.08.109
[Indexed for MEDLINE]
Free PMC Article

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