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This second edition compiles state-of-art protocols to study the molecular mechanisms that determine proteome composition and contribute to maintaining protein homeostasis. Chapters describe methods that range from the in vitro analysis of enzyme cascades to real-time in vivo studies. 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, Plant Proteostasis: Methods and Protocols, Second Edition aims to be a useful practical guide to researches to help further their study in this field.
List of contents
Observing real-time ubiquitination in high throughput with fluorescence polarization.- Identification and Characterization of Physiological Pairing of E2 Ubiquitin-Conjugating-Enzymes and E3 Ubiquitin Ligases.- Immunoprecipitation of Cullin Ring Ligases (CRLs) in Arabidopsis thaliana seedlings.- An in vitro assay to recapitulate hormone-triggered & SCF-mediated protein ubiquitylation.- Analysis of proteasome-associated ubiquitin ligase activity.- Measuring the DUB Activity of Arabidopsis Deubiquitylating enzymes.- SUMO conjugation and SUMO chain formation by plant enzymes.- Kinetic analysis of plant SUMO conjugation machinery.- Expression, purification, and enzymatic analysis of plant SUMO proteases.- Monitoring autophagic flux in the model single-celled microalga Chlamydomonas reinhardtii.- Detection of autophagy in plants by fluorescence microscopy.- Characterization of ATG8-family interactors by Isothermal Titration Calorimetry.- Protocols for studying protein stability in a Arabidopsis protoplast transient expression system.- Relative Protein Lifetime Measurement in Plants using Tandem Fluorescent Protein Timers.- Detection and Quantification of Protein Aggregates In Plants.- Using intrinsic fluorescence to measure protein stability upon thermal and chemical denaturation.- Purification and detection of ubiquitinated plant proteins using Tandem Ubiquitin Binding Entities.- Titanium oxide-based phosphopeptide enrichment from Arabidopsis seedlings.- Chloroplast Envelope Membrane Subfractionation from Arabidopsis and Pea.- Chromatin Enrichment for Proteomics in Plants (ChEP-P).- Proximity-dependent in vivo Biotin Labeling for Interactome Mapping in Marchantia polymorpha.- Tandem mass tag-based phospho-proteomics in plants.- Analysis of peptide hormone maturation and processing specificity using isotope-labeledpeptides.- Improved identification of protease cleavage sites by in-gel reductive dimethylation.- A Pipeline to Monitor Proteasome Homeostasis in Plants.- Bioinformatics tools for exploring the SUMO Gene Network, an update.- Coexpression network constriction and visualization from transcriptomes underlying ER stress responses.- Approaches for the identification of intrinsically disordered protein domains.
