Tandem affinity purification and mass spectrometric analysis of ubiquitylated proteins in Arabidopsis.

ABSTRACT: Protein ubiquitylation is a central regulatory mechanism that controls numerous processes in plants, including hormone signaling, developmental progression, responses to biotic and abiotic challenges, protein trafficking and chromatin structure. Despite data implicating thousands of plant proteins as targets, so far only a few have been conclusively shown to be ubiquitylated in planta. Here we describe a method to isolate ubiquitin-protein conjugates from Arabidopsis that exploits a stable transgenic line expressing a synthetic poly-UBQ gene encoding ubiquitin (Ub) monomers N-terminally tagged with hexahistidine. Following sequential enrichment by Ub-affinity and nickel chelate-affinity chromatography, the ubiquitylated proteins were trypsinized, separated by two-dimensional liquid chromat

SUBMITTER: Saracco SA 

PROVIDER: S-EPMC3639010 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature