Department of Experimental Neurodegeneration, University Medical Center Goettingen, Goettingen 37073, Germany.
Base Pharmaceuticals, Boston, MA 02129, USA.
STAR Protoc. 2020 Dec 10;1(3):100217. doi: 10.1016/j.xpro.2020.100217. eCollection 2020 Dec 18.
Photoconversion enables real-time labeling of protein sub-populations inside living cells, which can then be tracked with submicrometer resolution. Here, we detail the protocol of comparing protein dynamics inside membraneless organelles in live HEK293T cells using a CRISPR-Cas9 PABPC1-Dendra2 marker of stress granules. Measuring internal dynamics of membraneless organelles provides insight into their functional state, physical properties, and composition. Photoconversion has the advantage over other imaging techniques in that it is less phototoxic and allows for dual color tracking of proteins. For complete details on the use and execution of this protocol, please refer to Amen and Kaganovich (2020).
光转化使实时标记活细胞内的蛋白质亚群成为可能，随后可以亚微米分辨率对其进行追踪。在此，我们详细描述了使用 CRISPR-Cas9 PABPC1-Dendra2 应激颗粒标记物比较活 HEK293T 细胞中无膜细胞器内蛋白质动力学的方案。测量无膜细胞器的内部动力学可以深入了解其功能状态、物理特性和组成。与其他成像技术相比，光转化具有光毒性较小和允许双色追踪蛋白质的优势。有关该方案使用和执行的完整详细信息，请参阅 Amen 和 Kaganovich（2020 年）。
