Pull-down analysis of interactions among jasmonic acid core signaling proteins.

Pull-down assays are key tools to test specific protein-protein interactions and have been particularly fruitful in jasmonate signaling research. Here, we describe a standard protocol in which a matrix-bound "bait" protein, expressed in Escherichia coli, pulls down a "prey" protein that is soluble in a protein extract obtained from Arabidopsis thaliana plant tissues. The pulled-down protein can be detected by immunoblot with protein-specific or epitope-specific antibodies. Such a pull-down method was used to reveal interactions among components of the jasmonic acid signaling, including hormone-dependent coreceptor interaction, homodimerization and heterodimerization of JASMONATE ZIM DOMAIN repressors, and interactions among other corepressor components and with transcription factors. Pull-down assays contributed not only to shape this signaling pathway but also to identify the active jasmonate hormone.