Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, Germany.
Methods Mol Biol. 2022;2382:19-28. doi: 10.1007/978-1-0716-1744-1_2.
The cell cycle is a complex sequence of events by which cells grow and divide mitotically or meiotically. Mitosis results in the generation of two identical daughter cells, while meiosis generates gametes as a prerequisite for sexual reproduction. To study the localization and dynamics of proteins involved in the regulation and proceeding of the cell cycle, life cell imaging of proteins fused to fluorescent tags can be performed. However, in some cases this approach cannot be applied, e.g., due to low fluorescence intensity, fast bleaching, or degradation of recombinant proteins by the proteasome pathway. Instead, immunolabeling with protein-specific antibodies offers a useful approach for the analysis of intact cells. Alternatively, immunolabeling can also be applied to isolated and/or flow-sorted nuclei of particular cell cycle stages (G1, S, and G2) or of different endopolyploidy levels. The following chapter will detail indirect immunolabeling protocols to analyze the subcellular localization and distribution of cell cycle-specific proteins in Arabidopsis thaliana.
细胞周期是一个复杂的事件序列，细胞通过有丝分裂或减数分裂进行增殖和分裂。有丝分裂导致两个相同的子细胞的产生，而减数分裂产生配子作为有性繁殖的前提。为了研究参与细胞周期调控和进程的蛋白质的定位和动态，可对融合荧光标签的蛋白质进行活细胞成像。然而，在某些情况下，这种方法不能应用，例如，由于荧光强度低、快速漂白或重组蛋白被蛋白酶体途径降解。相反，用蛋白质特异性抗体进行免疫标记为分析完整细胞提供了一种有用的方法。或者，免疫标记也可以应用于特定细胞周期阶段（G1、S 和 G2）或不同的内多倍体水平的分离和/或流式分选的核。以下章节将详细介绍间接免疫标记方案，以分析拟南芥中细胞周期特异性蛋白质的亚细胞定位和分布。
