Fonseca Sandra, Solano Roberto
Departamento de Genética Molecular de Plantas, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
Methods Mol Biol. 2013;1011:159-71. doi: 10.1007/978-1-62703-414-2_13.
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.
下拉实验是检测特定蛋白质-蛋白质相互作用的关键工具，在茉莉酸信号转导研究中成果颇丰。在此，我们描述一种标准方案，其中在大肠杆菌中表达的与基质结合的“诱饵”蛋白可下拉一种可溶于拟南芥植物组织获得的蛋白质提取物中的“猎物”蛋白。通过用蛋白质特异性或表位特异性抗体进行免疫印迹可检测被下拉的蛋白。这种下拉方法被用于揭示茉莉酸信号转导组分之间的相互作用，包括激素依赖性共受体相互作用、茉莉酸ZIM结构域阻遏物的同二聚化和异二聚化，以及其他共阻遏物组分之间及其与转录因子的相互作用。下拉实验不仅有助于构建这一信号通路，还有助于鉴定活性茉莉酸激素。
