Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary.
Department of Solid State Physics, Faculty of Science and Technology, University of Debrecen, Debrecen, Hungary.
Curr Protoc. 2024 May;4(5):e1045. doi: 10.1002/cpz1.1045.
The cell surface distribution patterns (clustering) of membrane proteins have been widely investigated in cell biology. Here we describe a novel transmission electron microscopic (TEM) protocol designed to improve the quality of information obtained about the protein distribution patterns detected. This novel method makes it possible to study the clustering of all transmembrane proteins on one half of the cytoplasmic membrane of a whole cell. To achieve better imaging, we combine various methods, including critical-point drying, fixation of gold beads with a carbon layer, and a newly developed chemical thinning method. In addition, in our image-processing algorithm, we implemented pair correlation and pair cross-correlation functions, providing more details and better quantitative accuracy in characterizing the size and numbers of possible protein clusters. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Sample preparation and transmission electron micrography Alternate Protocol: Direct cell labeling for transmission electron micrography Basic Protocol 2: Analysis of TEM images to detect immunogold-labeled proteins.
细胞膜蛋白的表面分布模式（聚类）在细胞生物学中得到了广泛研究。在这里，我们描述了一种新的透射电子显微镜（TEM）方案，旨在提高有关检测到的蛋白质分布模式的信息质量。这种新方法使得研究整个细胞细胞质膜一半上所有跨膜蛋白的聚类成为可能。为了获得更好的成像效果，我们结合了各种方法，包括临界点干燥、带有碳层的金珠固定以及新开发的化学减薄方法。此外，在我们的图像处理算法中，我们实现了对相关函数和交叉相关函数的分析，从而在表征可能的蛋白质聚类的大小和数量方面提供了更详细和更准确的定量结果。© 2024 作者。Wiley Periodicals LLC 出版的《当代协议》。基本方案 1：样品制备和透射电子显微镜术 备选方案：用于透射电子显微镜术的直接细胞标记 基本方案 2：分析 TEM 图像以检测免疫金标记的蛋白质。
