Center for Advanced Imaging (CAi), Heinrich-Heine University, Düsseldorf, Germany.
University Library, University Duisburg-Essen, Duisburg, Germany.
Methods Mol Biol. 2022;2379:197-208. doi: 10.1007/978-1-0716-1791-5_12.
Protein-protein interactions in living plant cells can be measured by changes in fluorescence anisotropy due to homo-FRET (Förster Resonance Energy Transfer). Here, the energy transfer between identical fluorophores, e.g., enhanced green fluorescent protein (EGFP) fused to a protein of interest, serves as a read-out for protein interaction and clustering. By applying homo-FRET imaging, not only dimeric complexes, but also bigger homomeric complex formation can be followed in vivo at high spatial and temporal resolution. Therefore, this method provides a powerful tool to investigate changes in complex formation over time in their natural environment with high precision at a subcellular level. Here, we describe the necessary theoretical background and how homo-FRET imaging is practically carried out. We also discuss potential pitfalls and points of consideration.
活体细胞内的蛋白质-蛋白质相互作用可以通过荧光各向异性的变化来测量，这种变化是由于同型 FRET（Förster 共振能量转移）引起的。在这里，相同荧光团（例如，融合到感兴趣的蛋白质上的增强型绿色荧光蛋白（EGFP））之间的能量转移可作为蛋白质相互作用和聚集的读出信号。通过应用同型 FRET 成像，可以在高时空分辨率下在体内跟踪不仅是二聚体复合物，而且还有更大的同型复合物的形成。因此，该方法提供了一种强大的工具，可以在亚细胞水平上以高精度研究其天然环境中复合物形成随时间的变化。在这里，我们描述了必要的理论背景以及如何实际进行同型 FRET 成像。我们还讨论了潜在的陷阱和注意点。
