Institut de Recherches Cliniques de Montréal, 110 Avenue des Pins Ouest, Montréal, QC H2W 1R7, Canada; Département de Biochimie et Médecine Moléculaire, Faculté de Médecine, Université de Montréal, Montréal, QC H3T 1J4, Canada.
Département de Biochimie et Médecine Moléculaire, Faculté de Médecine, Université de Montréal, Montréal, QC H3T 1J4, Canada.
STAR Protoc. 2023 Sep 15;4(3):102359. doi: 10.1016/j.xpro.2023.102359. Epub 2023 Jun 15.
Two isoforms of the nuclear pore complex (NPC) have been identified in the yeast S. cerevisiae, which coexist at the periphery of the nucleus and differ by the presence or absence of a nuclear basket. Here, we present a protocol to isolate the two types of NPCs from the same cell extract and dissect their interactomes. We describe steps for powder preparation and magnetic bead conjunction and detail differential affinity purification and outcome evaluation through SDS-PAGE, silver staining, and mass spectrometry analysis. For complete details on the use and execution of this protocol, please refer to Bensidoun et al..
已在酵母 S. cerevisiae 中鉴定出核孔复合体 (NPC) 的两种同工型，它们共存于核的外周，区别在于是否存在核篮。在此，我们提供了一种从同一细胞提取物中分离两种类型 NPC 并剖析其相互作用组的方案。我们描述了粉末制备和磁珠偶联的步骤，并详细说明了通过 SDS-PAGE、银染和质谱分析进行差异亲和纯化和结果评估的步骤。有关此方案使用和执行的完整详细信息，请参阅 Bensidoun 等人的研究。
