Investigating mitotic spindle assembly and function in vitro using Xenopus laevis egg extracts
Investigating mitotic spindle assembly and function in vitro using Xenopus laevis egg extracts
复制标题
DOI：
10.1038/nprot.2006.396
复制
发表时间：
2006-01-01
期刊：
影响因子：
14.8
通讯作者：
Heald, Rebecca
中科院分区：
文献类型：
--
作者：
Hannak, Eva;Heald, Rebecca
非洲爪蟾卵提取液为体外细胞分裂过程的研究提供了一个强有力的系统，通过生化重建和操作，以及显微镜分析。我们提供了中期阻滞提取物的制备方案和体外检测纺锤体组装的以下途径：1)精子核添加到减数分裂提取物中，支持未复制染色体周围半纺锤体和双纺锤体结构的形成；2)精子细胞核加入到提取物中，经过间期循环，形成纺锤体，能够经历后期和染色体分离；3)在染色质包裹的小珠周围形成纺锤体。最后，我们描述了通过免疫消耗或添加抑制剂（如显性阴性结构）来抑制特定蛋白质的方法。这些技术可用于分析特定蛋白质的有丝分裂功能。提取液的制备大约需要1.5小时，纺锤体组装实验需要1 - 3小时，如果进行免疫消耗，则需要额外的1 - 3小时。
Extracts from Xenopus laevis eggs provide a powerful system for the study of cell division processes in vitro through biochemical reconstitution and manipulation, and microscopic analysis. We provide protocols for the preparation of metaphase-arrested extracts and in vitro assays to examine the following pathways of spindle assembly: 1) Sperm nuclei added to meiotic extracts, supporting the formation of half-spindles and bipolar spindle structures around unreplicated chromosomes; 2) sperm nuclei added to extracts that cycle through interphase and form spindles that are capable of undergoing anaphase and chromosome segregation; and 3) spindle formation around chromatin-coated beads. Finally, we describe methods to inhibit a specific protein by immunodepletion or addition of an inhibitor such as a dominant-negative construct. These techniques can be used to analyze the mitotic function of a given protein. It takes similar to 1.5 h to prepare the extract, 1 - 3 h for spindle-assembly experiments and an additional 1 - 3 h if immunodepletion is performed.