title: Immunoprecipitation of Cullin-RING Ligases (CRLs) in Arabidopsis thaliana Seedlings.
abstract: CRL (Cullin-RING ubiquitin ligase) is the major class of plant E3 ubiquitin ligases. Immunoprecipitation-based methods are useful techniques for revealing interactions among Cullin-RING Ligase (CRL) subunits or between CRLs and other proteins, as well as for detecting poly-ubiquitin modifications of the CRLs themselves. Here, we describe two immunoprecipitation (IP) procedures suitable for CRLs in Arabidopsis: a procedure for IP analysis of CRL subunits and their interactors and a second procedure for in vivo ubiquitination analysis of the CRLs. Both protocols can be divided into two major steps: (1) preparation of cell extracts without disruption of protein interactions and (2) affinity purification of the protein complexes and subsequent detection. We provide a thorough description of all the steps, as well as advice on how to choose proper buffers for these analyses. We also suggest a series of negative controls that can be used to verify the specificity of the procedure.
authors.affiliation: RIKEN Plant Science Center, Tsurumi, Yokohama, Kanagawa, 230-0045, Japan.
authors.forename: Anna
authors.initials: A
authors.lastname: Franciosini
authors.affiliation: Dipartimento di Biologia e Biotecnologie "C. Darwin", Sapienza Università di Roma, Piazzale Aldo Moro 5, 00185, Rome, Italy. giovanna.serino@uniroma1.it.
authors.forename: Giovanna
authors.initials: G
authors.lastname: Serino
mesh_terms: D017360:Arabidopsis / Q000201:enzymology; D044842:Cullin Proteins / Q000737:chemistry / Q000302:isolation & purification*; D008967:Molecular Biology / Q000379:methods*; D036226:Seedlings / Q000737:chemistry / Q000201:enzymology; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry*; D054875:Ubiquitination
chemical_list: D044842:Cullin Proteins; D044767:Ubiquitin-Protein Ligases
keywords: Coimmunoprecipitation; Cullin-RING ubiquitin ligase; Immunoblot; Immunoprecipitation; Ubiquitin