Read more
This volume details novel and popular approaches to decipher the Ubiquitin Code. Chapters guide readers through ubiquitin chain complexity, methods to generate non-hydrolysable ubiquitin-NEDD8, a fully synthetic method for the linear synthesis of fluorescently labelled rhodamine LC3A and LC3B, a HECT ligase-mediated ubiquitylation in vitro, a RNAi-based screening to identify DUBs, a Yuh1 activity assay, a pull-down to isolate K48 and K63 chains using specific nanobodies, identification of NeDDylated protein based in a NEDD8R74K mutant, in vivo identification of NEDDylated proteins from liver diseases, UbL-ID method to identify ubiquitin-like modified proteins, a LC3/GABARAP capturing system, and methods to isolate peptides that are closely associated with proteasomes or trapped inside the core particle of proteasomes in eukaryotic cells, among others. Written in the successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls.
Authoritative and cutting-edge, The Ubiquitin Code aims to to be a useful practical guide to researches to help further their study in this field.
List of contents
Thioester and oxyester linkages in the ubiquitin system.- Getting to the root of branched ubiquitin chains: A review of current methods and functions.- Chemical synthesis of non-hydrolysable Ubiquitin(-like) hybrid chains.- Total Linear Chemical Synthesis of LC3A and LC3B.- A microbead-based flow cytometry assay to assess the activity of Ubiquitin and Ubiquitin-like conjugating enzymes.- Monitoring HECT ubiquitination activity in vitro.- RNAi-based screening for the identification of specific substrate-deubiquitinase pairs.- Strategies for monitoring 'ubiquitin C-terminal hydrolase 1' (Yuh1) activity.- Isolation and Mass Spectrometry Identification of K48 and K63 Ubiquitin Proteome using Chain-Specific Nanobodies.- A mass spectrometry-based strategy for mapping modification sites for the Ubiquitin-like modifier NEDD8.- Isolation of the hepatic ubiquitome/NEDDylome by streptavidin pull-down assay in the biotinylated ubiquitin (bioUb)/biotinylated NEDD8 (bioNEDD8) transgenic mice.- The Lambda display technology: a useful tool for the identification of ubiquitin and ubiquitin like-binding domains.- SUMO-ID: a strategy for the identification of SUMO-dependent proximal interactors.- Analysis of ATG8 family members using LC3-Interacting Regions (LIR)-based molecular traps.- A computational tool for analysis of mass spectrometry data of ubiquitin enriched samples.- In-plate and in-gel assays for the assessment of proteasome activity in Caenorhabditis elegans.- Isolation of Proteasome-Trapped Peptides (PTPs) for degradome analysis.
