Institute for Research in Immunology and Cancer, Université de Montréal, Montréal, Québec H3T 1J4, Canada.
Molecular Biology Program, Université de Montréal, Montréal, Québec H3C3J7, Canada.
J Proteome Res. 2023 Aug 4;22(8):2765-2773. doi: 10.1021/acs.jproteome.3c00266. Epub 2023 Jul 18.
Current protocols used to extract and purify histones are notoriously tedious, especially when using yeast cells. Here, we describe the use of a simple filter-aided sample preparation approach enabling histone extraction from yeast and mammalian cells using acidified ethanol, which not only improves extraction but also inactivates histone-modifying enzymes. We show that our improved method prevents N-terminal clipping of H3, an artifact frequently observed in yeast cells using standard histone extraction protocols. Our method is scalable and provides efficient recovery of histones when extracts are prepared from as few as two million yeast cells. We further demonstrate the application of this approach for the analysis of histone modifications in fungal clinical isolates available in a limited quantity. Compared with standard protocols, our method enables the study of histones and their modifications in a faster, simpler, and more robust manner.
目前用于提取和纯化组蛋白的方法非常繁琐，尤其是在使用酵母细胞时。在这里，我们描述了一种简单的过滤辅助样品制备方法，该方法可使用酸化乙醇从酵母和哺乳动物细胞中提取组蛋白，不仅可提高提取效率，还可使组蛋白修饰酶失活。我们发现，我们改进的方法可防止 H3 的 N 端截断，这是使用标准组蛋白提取方案在酵母细胞中经常观察到的一种假象。我们的方法具有可扩展性，当从少至 200 万个酵母细胞制备提取物时，可有效回收组蛋白。我们进一步证明了该方法在分析数量有限的真菌临床分离株中的组蛋白修饰方面的应用。与标准方法相比，我们的方法可更快速、更简单、更稳健地研究组蛋白及其修饰。
