Institute of Biomedical Research, Yunnan University, Kunming 650500, China.
State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Institute of Stem Cells and Regeneration, Chinese Academy of Sciences, Beijing 100101, China.
STAR Protoc. 2022 Dec 16;3(4):101709. doi: 10.1016/j.xpro.2022.101709. Epub 2022 Sep 21.
Here, we present a general protocol for mimicking lipid-mediated phase separation on the membrane using giant unilamellar vesicles (GUVs). In this protocol, we use GUVs to mimic Ago1 protein's phase separation behavior on the membrane through binding with phosphoinositides (PIPs). We provide procedures to prepare fluorescent-labeled Ago1 protein and PI(4,5)P2-containing GUVs, followed by steps to assess Ago1 protein's phase separation in 3D time-lapse images. This protocol can be applied to investigate a membrane-associated protein's behavior on the membrane. For complete details on the use and execution of this protocol, please refer to Gao et al. (2022).
在这里，我们提出了一个使用巨大的单层囊泡（GUV）在膜上模拟脂质介导的相分离的一般方案。在这个方案中，我们使用 GUV 来模拟 Ago1 蛋白通过与磷酸肌醇（PIPs）结合在膜上的相分离行为。我们提供了制备荧光标记的 Ago1 蛋白和含有 PI(4,5)P2 的 GUV 的程序，然后介绍了评估 Ago1 蛋白在 3D 延时图像中的相分离的步骤。这个方案可以用来研究膜相关蛋白在膜上的行为。有关此方案的使用和执行的完整详细信息，请参阅 Gao 等人。（2022）。
