Liu Xiao, Tang Yu
State Key Laboratory of Wheat Improvement, Peking University Institute of Advanced Agricultural Sciences, Shandong Laboratory of Advanced Agricultural Sciences at Weifang, Weifang, Shandong, China.
Methods Mol Biol. 2025;2958:17-34. doi: 10.1007/978-1-0716-4714-1_2.
The nucleus represents a fundamental feature shared by eukaryotic cells and enclosed by a highly regulated membrane barrier, the nuclear envelope (NE). Despite numerous studies elucidating the functional significance and diversity of the NE in animals and yeast, comprehensive knowledge regarding its intricate composition and functions in plants remains limited. In this chapter, we describe subtractive proteomic approach to profile novel NE components with the model organism Arabidopsis, including detailed methods for isolation of nuclei using Percoll density gradients, extraction of nuclear membranes, isolation of endoplasmic reticular enriched microsomal membranes, determination of the proteome, and validation of the enrichment at nuclear periphery. Additionally, this approach is applicable for identification of nuclear envelope protein components from diverse tissues or other plant species.
细胞核是真核细胞共有的基本特征，由高度调控的膜屏障——核膜（NE）包裹。尽管有大量研究阐明了动物和酵母中核膜的功能意义和多样性，但关于其在植物中复杂组成和功能的全面知识仍然有限。在本章中，我们描述了用模式生物拟南芥进行消减蛋白质组学方法来分析新的核膜成分，包括使用Percoll密度梯度分离细胞核、提取核膜、分离富含内质网的微粒体膜、蛋白质组测定以及核周富集验证的详细方法。此外，该方法适用于从不同组织或其他植物物种中鉴定核膜蛋白成分。
