通过对活的U-2 OS细胞进行透化处理和酶处理来确定蛋白质和RNA对凝聚物组织贡献的实验方案。
Protocol for determining the contribution of protein and RNA to condensate organization by permeabilizing and enzyme-treating live U-2 OS cells.
作者信息
Parker Dylan M, Parker Roy
机构信息
Department of Biochemistry, University of Colorado Boulder, Boulder, CO 80309, USA; Howard Hughes Medical Institute, University of Colorado Boulder, Boulder, CO 80309, USA.
Department of Biochemistry, University of Colorado Boulder, Boulder, CO 80309, USA; Howard Hughes Medical Institute, University of Colorado Boulder, Boulder, CO 80309, USA.
出版信息
STAR Protoc. 2025 Apr 9;6(2):103744. doi: 10.1016/j.xpro.2025.103744.
Ribonucleoprotein granules are comprised of protein and RNA. We present a protocol for querying the relative contribution of protein and RNA interactions to granule organization by permeabilizing and treating cells with proteinase or RNase enzymes. We then detail steps for performing single-molecule fluorescence in situ hybridization (smFISH) on enzyme-treated samples for RNA visualization. Finally, we describe detailed instructions for quantifying results generated with this protocol. This protocol can potentially query the contribution of protein-RNA, protein-protein, and RNA-RNA interactions to the organization of any intracellular granule. For complete details on the use and execution of this protocol, refer to Parker et al..
核糖核蛋白颗粒由蛋白质和RNA组成。我们提供了一种方案，通过用蛋白酶或核糖核酸酶通透处理细胞，来探究蛋白质与RNA相互作用对颗粒组织的相对贡献。然后，我们详细说明了对酶处理后的样品进行单分子荧光原位杂交（smFISH）以实现RNA可视化的步骤。最后，我们描述了对该方案产生的结果进行定量的详细说明。该方案有可能探究蛋白质-RNA、蛋白质-蛋白质和RNA-RNA相互作用对任何细胞内颗粒组织的贡献。有关该方案使用和执行的完整详细信息，请参考帕克等人的研究。
Zotero 插件