D'Avino Pier Paolo, Archambault Vincent, Przewloka Marcin R, Zhang Wei, Laue Ernest D, Glover David M
Cancer Research UK Cell Cycle Genetics Research Group, Department of Genetics, University of Cambridge, Cambridge, UK.
Methods Mol Biol. 2009;545:99-112. doi: 10.1007/978-1-60327-993-2_6.
The identification of all the individual components that constitute the plethora of complexes in each cell type represents perhaps the most exciting challenge of postgenomic biology. This is particularly important in the study of events such as mitosis and cytokinesis, in which rapid and precise protein-protein interactions regulate both the direction and accuracy of these intricate processes. Here we describe an experimental strategy to isolate protein complexes involved in mitosis and cytokinesis in cultured Drosophila cells. This method involves the tagging of the bait protein with two IgG binding domains of Protein A and the isolation of the tagged bait along with its interacting partners by a single affinity purification step. These isolated complexes can then be analysed by several methods including mass spectrometry and Western blotting. Although this method has proven very successful in isolating mitotic and cytokinetic complexes, it can also be used to characterise protein complexes involved in many other cellular processes.
确定构成每种细胞类型中大量复合物的所有单个成分，可能是后基因组生物学最令人兴奋的挑战。这在有丝分裂和胞质分裂等事件的研究中尤为重要，在这些事件中，快速而精确的蛋白质-蛋白质相互作用调节着这些复杂过程的方向和准确性。在这里，我们描述了一种实验策略，用于在培养的果蝇细胞中分离参与有丝分裂和胞质分裂的蛋白质复合物。该方法包括用蛋白A的两个IgG结合结构域标记诱饵蛋白，并通过单一亲和纯化步骤分离标记的诱饵及其相互作用伙伴。然后可以通过包括质谱分析和蛋白质印迹在内的几种方法对这些分离的复合物进行分析。尽管该方法已被证明在分离有丝分裂和胞质分裂复合物方面非常成功，但它也可用于表征参与许多其他细胞过程的蛋白质复合物。
