Durhan Seyda Tugce, Sezer Enise Nalli, Son Cagdas Devrim, Baloglu Fatma Kucuk
Department of Biological Sciences, 52984Middle East Technical University, Ankara, Turkey.
Department of Biology, 187438Giresun University, Giresun, Turkey.
Appl Spectrosc. 2023 Mar;77(3):292-302. doi: 10.1177/00037028221140914. Epub 2022 Nov 25.
Protein-protein interactions (PPIs) have great importance for intracellular signal transduction and sustaining the homeostasis of an organism. Thus, the identification of PPIs is necessary to better understand the downstream signaling functions of the proteins in healthy and pathological conditions. Förster resonance energy transfer (FRET) between fluorescent proteins (FPs) is a powerful tool for detecting PPIs in living cells. In literature, FRET analysis methods such as donor photobleaching (FLIM), acceptor photobleaching, spectral imaging, and the three-filter cube method (sensitized emission) are abundantly applied to investigate PPIs; however, they require various expensive instrumentations, and their calculation methods are very time consuming. Since confocal microscopy applications and live cell-based techniques of FRET are very costly, scientists sometimes prefer plate readers for FRET experiments. However, plate reader applications also have many disadvantages and considerations compared to confocal fluorescence microscopy, and complex calculation procedures should be performed. To overcome these problems, we propose a FRET-based high-throughput assay method with a standard monochromator-based microplate reader, which is generally available in most biochemistry laboratories, and an alternative calculation procedure. This rapid, low cost, and effective analysis method enables the scientists to prescreen PPIs in living cells as a preliminary study and quick glance at the experiment before preparing the whole experimental setup with the expensive instrumentations. Additionally, the alternative calculation procedure provides the FRET area comparison without complex bleed-through calculations in a non-conventional manner by shortening the analysis processes with this quick and uncomplicated spectral representation.
蛋白质-蛋白质相互作用（PPIs）对于细胞内信号转导和维持生物体的稳态具有重要意义。因此，识别PPIs对于更好地理解蛋白质在健康和病理条件下的下游信号功能是必要的。荧光蛋白（FPs）之间的荧光共振能量转移（FRET）是检测活细胞中PPIs的有力工具。在文献中，诸如供体光漂白（荧光寿命成像）、受体光漂白、光谱成像和三滤光片法（敏化发射）等FRET分析方法被大量应用于研究PPIs；然而，它们需要各种昂贵的仪器设备，并且其计算方法非常耗时。由于共聚焦显微镜应用和基于活细胞的FRET技术成本非常高，科学家有时会选择酶标仪进行FRET实验。然而，与共聚焦荧光显微镜相比，酶标仪应用也有许多缺点和需要考虑的因素，并且需要执行复杂的计算程序。为了克服这些问题，我们提出了一种基于FRET的高通量检测方法，该方法使用大多数生物化学实验室普遍可用的基于标准单色仪的酶标仪以及一种替代计算程序。这种快速、低成本且有效的分析方法使科学家能够在活细胞中预先筛选PPIs作为初步研究，并在使用昂贵仪器设备准备整个实验装置之前快速浏览实验。此外，这种替代计算程序通过以这种快速且简单的光谱表示缩短分析过程，以非传统方式在无需复杂的渗漏计算的情况下提供FRET面积比较。
