Institute of Chemical Biology, Department of Chemistry, Imperial College London, South Kensington Campus, London, UK.
J Biophotonics. 2013 May;6(5):398-408. doi: 10.1002/jbio.201200185. Epub 2012 Nov 26.
Fluorescence lifetime measurements can provide quantitative readouts of local fluorophore environment and can be applied to biomolecular interactions via Förster resonant energy transfer (FRET). Fluorescence lifetime imaging (FLIM) can therefore provide a high content analysis (HCA) modality to map protein-protein interactions (PPIs) with applications in drug discovery, systems biology and basic research. We present here an automated multiwell plate reader able to perform rapid unsupervised optically sectioned FLIM of fixed and live biological samples and illustrate its potential to assay PPIs through application to Gag protein aggregation during the HIV life cycle. We demonstrate both hetero-FRET and homo-FRET readouts of protein aggregation and report the first quantitative evaluation of a FLIM HCA assay by generating dose response curves through addition of an inhibitor of Gag myristoylation. Z' factors exceeding 0.6 are realised for this FLIM FRET assay.
荧光寿命测量可以提供局部荧光团环境的定量读数，并可通过Förster 共振能量转移（FRET）应用于生物分子相互作用。因此，荧光寿命成像（FLIM）可以提供高内涵分析（HCA）模式，以绘制蛋白质-蛋白质相互作用（PPIs）图谱，应用于药物发现、系统生物学和基础研究。我们在这里介绍一种能够对固定和活生物样品进行快速非监督光学切片 FLIM 的自动化多微孔板读取器，并通过将其应用于 HIV 生命周期中的 Gag 蛋白聚集来说明其测定 PPIs 的潜力。我们展示了蛋白质聚集的异 FRET 和同 FRET 读出，并通过添加 Gag 豆蔻酰化抑制剂来生成剂量反应曲线，首次对 FLIM HCA 测定进行了定量评估。该 FLIM FRET 测定的 Z' 因子超过 0.6。
