Yu Shenliang
Department of Physiology, University of California, San Francisco, San Francisco, CA, USA.
Methods Mol Biol. 2025;2888:193-200. doi: 10.1007/978-1-0716-4318-1_13.
This chapter describes two imaging-based approaches for examining the localization of bridge-like lipid transfer proteins at membrane contact sites during native biological processes. These approaches use multi-color fluorescence imaging, enabling high spatial and temporal resolution and overcoming the limitations of biochemical methods. The first approach involves immunofluorescence in fixed cells, while the second utilizes time-lapse imaging in live cells. These methods are showcased through the example of ATG2, an essential autophagy-related protein, and demonstrate the ability to overcome technical difficulties such as large protein size, lack of high-quality antibodies, and imaging highly dynamic subcellular structures. These described methods provide a powerful tool for understanding protein function and biological processes and can be widely applied to various research questions in cell biology.
本章介绍了两种基于成像的方法，用于在天然生物学过程中检查桥状脂质转移蛋白在膜接触位点的定位。这些方法使用多色荧光成像，能够实现高空间和时间分辨率，并克服了生化方法的局限性。第一种方法涉及固定细胞中的免疫荧光，而第二种方法则利用活细胞中的延时成像。通过自噬相关必需蛋白ATG2的例子展示了这些方法，并证明了它们有能力克服诸如蛋白质尺寸大、缺乏高质量抗体以及对高度动态的亚细胞结构进行成像等技术难题。这些所描述的方法为理解蛋白质功能和生物学过程提供了一个强大的工具，并且可以广泛应用于细胞生物学中的各种研究问题。
