Fischell Department of Bioengineering, University of Maryland, College Park, MD, USA.
Program in Molecular and Cell Biology, University of Maryland, College Park, MD, USA.
Methods Mol Biol. 2024;2843:15-23. doi: 10.1007/978-1-0716-4055-5_2.
Bacterial extracellular vesicles (BEVs) have emerged as mediators of transkingdom communication with numerous potential biotechnological applications. As such, investigation of BEV's protein composition holds promise to uncover new biological mechanisms, such as in microbiome-host communication or pathogen infection. Additionally, bioengineering of BEV protein composition can enhance their therapeutic potential. However, accurate assessment of BEV protein cargo is limited by their nanometer size, which precludes light microscopy imaging, as well as by co-isolation of protein impurities during separation processes. A solution to these challenges is found in immunogold transmission electron microscopy (TEM), which combines antibody-based labeling with direct visualization of BEVs. Several challenges are commonly encountered during immunogold TEM analysis of BEVs, most notably inefficient antibody labeling and poor contrast. Here, we present an optimized protocol for immunogold TEM analysis of BEVs that overcomes such challenges.
细菌细胞外囊泡 (BEV) 已成为跨物种交流的介质，具有众多潜在的生物技术应用。因此，研究 BEV 的蛋白质组成有望揭示新的生物学机制，例如微生物组-宿主通讯或病原体感染。此外，BEV 蛋白质组成的生物工程可以增强其治疗潜力。然而，由于 BEV 的纳米尺寸，其蛋白质货物的准确评估受到限制，这排除了光显微镜成像，并且在分离过程中也会共同分离出蛋白质杂质。免疫金透射电子显微镜 (TEM) 为这些挑战提供了一种解决方案，它将基于抗体的标记与 BEV 的直接可视化相结合。在 BEV 的免疫金 TEM 分析中，通常会遇到几个挑战，最明显的是抗体标记效率低下和对比度差。在这里，我们提出了一种优化的免疫金 TEM 分析 BEV 的方案，该方案克服了这些挑战。
