Generation of Monoubiquitin and K63-Linked Polyubiquitin Chains for Protein Interaction Studies

Abstract

Ubiquitylation is a posttranslational modification that utilizes protein-protein binding interactions to regulate cellular processes. In ubiquitin signaling, a vast array of mono- and polyubiquitin modifications to substrate proteins are recognized by a diverse group of ubiquitin-binding proteins. Identifying ubiquitin-binding proteins and characterizing their binding properties is necessary for understanding the structural basis of ubiquitin signaling. This chapter provides a means of studying ubiquitin-binding interactions in vitro by describing how to generate monoubiquitin and K63-linked polyubiquitin chains and perform pull-down assays with ubiquitin-binding proteins, which is of particular relevance for DNA damage and other signaling pathways.

Keywords: Cellular signaling; Polyubiquitin chains; Pull-down assay; Ubiquitin; Ubiquitin-activating enzyme; Ubiquitin-binding protein; Ubiquitin-conjugating enzyme; Ubiquitin-conjugation reaction.