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.
Highlights
The discovery of ubiquitin-mediated proteolysis [1] was one of the most seminal papers published in Biochemical and Biophysical Research Communications (BBRC), which won Aaron Ciechanover and Avram Herschko the Nobel Prize for Chemistry 26 years later
IAA or NEM have typically been included at concentrations of 5e10 mM in many publications, we find that up to 10-fold higher concentrations are needed to preserve the ubiquitylation status of some proteins (e.g. Interleukin receptor associated kinase-1 (IRAK1) (Fig. 2A) and ubiquitin chains (Fig. 2B)
An unambiguous way to demonstrate the covalent attachment of pUb chains to proteins is to capture the ubiquitylated form of the protein of interest (POI) from cell extracts using immobilised ubiquitin-binding domains (UBDs) of defined ubiquitin chain specificity (Table 1) followed by immunoblotting with antibodies raised against the POI
Summary
The discovery of ubiquitin-mediated proteolysis [1] was one of the most seminal papers published in Biochemical and Biophysical Research Communications (BBRC), which won Aaron Ciechanover and Avram Herschko the Nobel Prize for Chemistry 26 years later. Cellular processes and, to date, eight different types of ubiquitin chain linkage have been identified in cells [2,3]. These linkages are formed by the covalent attachment of the C-terminus of ubiquitin to the ε-amino groups of any of the seven lysine (K) residues in ubiquitin (K6, K11, K27, K29, K33, K48 and K63) or the a-amino group of its N-terminal methionine (M1) residue. We introduce a number of recommendations about how to optimise the quality of the information that can be obtained from such experiments, based on our own experiences and other published papers in the literature
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