Surface Spreading and Immunostaining of Yeast Chromosomes
Surface Spreading and Immunostaining of Yeast Chromosomes
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DOI：
10.3791/53081
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发表时间：
2015-08-01
影响因子：
1.2
通讯作者：
Bishop, Douglas K.
中科院分区：
文献类型：
--
作者：
Grubb, Jennifer;Brown, M. Scott;Bishop, Douglas K.
芽殖酵母酿酒酵母的细胞核尺寸较小，限制了光学显微镜在分析染色质结合蛋白的亚核分布方面的实用性。酵母细胞核的表面扩散导致染色质扩张，而不会损失结合的蛋白质。一种表面铺展方法平衡 DNA 结合蛋白的固定与去污剂处理。所演示的方法与 Josef Loidl 和 Franz Klein (1,2) 描述的方法略有修改。该方法已用于表征许多染色质结合蛋白在有丝分裂细胞周期各个阶段的定位，但对于研究减数分裂染色体结构（例如减数分裂重组体和联会复合体）特别有用。我们还描述了一种不需要使用Lipsol（一种专有洗涤剂）的改进，这在原始程序中是需要的，但不再在商业上可用。还描述了与染色体扩散方法兼容的免疫染色方案。
The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution of chromatin-bound proteins. Surface spreading of yeast nuclei results in expansion of chromatin without loss of bound proteins. A method for surface spreading balances fixation of DNA bound proteins with detergent treatment. The method demonstrated is slightly modified from that described by Josef Loidl and Franz Klein(1,2). The method has been used to characterize the localization of many chromatin-bound proteins at various stages of the mitotic cell cycle, but is especially useful for the study of meiotic chromosome structures such as meiotic recombinosomes and the synaptonemal complex. We also describe a modification that does not require use of Lipsol, a proprietary detergent, which was called for in the original procedure, but no longer commercially available. An immunostaining protocol that is compatible with the chromosome spreading method is also described.