Sidney Kimmel Comprehensive Cancer Center, Department of Radiation Oncology and Molecular Radiation Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA.
Proteomics. 2012 Oct;12(19-20):3044-8. doi: 10.1002/pmic.201200071. Epub 2012 Sep 10.
Efficient extraction and accurate quantification of nucleolar macromolecules are critical for in vitro analysis, especially for studying RNA, DNA, and protein dynamics under identical conditions. There is presently no single method that efficiently and simultaneously isolates these three macromolecular constituents from purified nucleoli. We have developed an optimized method, which without evident loss, extracts, and solubilizes protein recovered from a single sample following TRIzol isolation of RNA and DNA. The solubilized protein can be accurately quantified by protein bicinchoninic acid assay and assessed by polyacrylamide gel electrophoresis. We have successfully applied this approach to extract and quantify all three nucleolar components, and to study nucleolar protein responses after actinomycin D treatment.
高效提取和准确定量核仁大分子对于体外分析至关重要，特别是在相同条件下研究 RNA、DNA 和蛋白质动态变化时。目前还没有一种单一的方法可以有效地同时从纯化的核仁中分离这三种大分子成分。我们开发了一种优化的方法，该方法在 TRIzol 分离 RNA 和 DNA 后，无需明显损失即可从单个样品中提取和溶解蛋白质。溶解的蛋白质可以通过蛋白质双缩脲比色法准确定量，并通过聚丙烯酰胺凝胶电泳进行评估。我们已经成功地应用这种方法提取和定量了所有三种核仁成分，并研究了放线菌素 D 处理后核仁蛋白的反应。
