Quantitative Assessment of Histone H2B Monoubiquitination in Yeast Using Immunoblotting.
Quantitative Assessment of Histone H2B Monoubiquitination in Yeast Using Immunoblotting.
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DOI：
10.3390/mps5050074
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发表时间：
2022-09-24
影响因子：
2.4
通讯作者：
Chandrasekharan, Mahesh B
中科院分区：
文献类型：
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作者：
Leng, Andrew M;Radmall, Kaitlin S;Shukla, Prakash K;Chandrasekharan, Mahesh B
对酿酒酵母和pombe Schizosaccharomyces酿酒酵母的研究增强了我们对组蛋白H2B单泛素化（H2Bub1）的调控和功能的认识，这是一个关键的表观遗传标记，在转录和其他过程中起重要作用。利用免疫印迹法检测酵母中的H2Bub1，已经极大地促进了针对酵母组蛋白H2B抗体的商业可用性和针对单泛素化人H2BK120抗体的交叉反应性。这些抗体不需要表达表位标记的组蛋白H2B来检测酵母中的H2Bub1。在这里，我们提供了一步一步的方案和最佳实践，用于酵母系统中H2Bub1的定量，从细胞提取物制备到使用市售抗体的免疫印迹。我们证明了商业抗体可以有效准确地检测酿酒葡萄球菌和pombe葡萄球菌中的H2Bub1。此外，我们发现组蛋白H2B的c端表位标记改变了酵母系统中H2Bub1的稳态水平。我们报告了一种切片印迹探测方法，结合连续稀释蛋白裂解物和使用可逆染色蛋白作为加载对照，共同为酵母中H2Bub1的定量评估提供了一种经济有效且敏感的方法。
Studies in Saccharomyces cerevisiae and Schizosaccharomyces pombe have enhanced our understanding of the regulation and functions of histone H2B monoubiquitination (H2Bub1), a key epigenetic marker with important roles in transcription and other processes. The detection of H2Bub1 in yeasts using immunoblotting has been greatly facilitated by the commercial availability of antibodies against yeast histone H2B and the cross-reactivity of an antibody raised against monoubiquitinated human H2BK120. These antibodies have obviated the need to express epitope-tagged histone H2B to detect H2Bub1 in yeasts. Here, we provide a step-by-step protocol and best practices for the quantification of H2Bub1 in yeast systems, from cell extract preparation to immunoblotting using the commercially available antibodies. We demonstrate that the commercial antibodies can effectively and accurately detect H2Bub1 in S. cerevisiae and S. pombe. Further, we show that the C-terminal epitope-tagging of histone H2B alters the steady-state levels of H2Bub1 in yeast systems. We report a sectioned blot probing approach combined with the serial dilution of protein lysates and the use of reversibly stained proteins as loading controls that together provide a cost-effective and sensitive method for the quantitative evaluation of H2Bub1 in yeast.