2-PHOTON FRET OF GFP-FUSION PROTEINS IN LIVING CELLS AND TISSUES

活细胞和组织中 GFP 融合蛋白的 2 光子干涉

• 批准号: 7365629
• 负责人: VICKIE J LAMORTE
• 金额: $ 0.39万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7365629
• 关键词: PHOTON; FRET; GFP; FUSION; PROTEINS

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We have developed a spectroscopic approach for monitoring fluorescence resonance energy transfer (FRET) in living cells. This method provides excellent spectral separation of GFP mutant signals within a subcellular imaging volume using two-photon excited fluorescence imaging and spectroscopy (TPIS-FRET). In contrast to current FRET-based methodologies, TPIS-FRET does not rely on the selection of optical filters, ratiometric image analysis, or bleedthrough correction algorithms. Inherent to the optical sectioning capabilities of TPIS-FRET, protein-protein interactions can be identified within discrete subcellular domains. To illustrate the applicability of this technique to the detection of homodimer formation, we demonstrated the in vivo association of promyleocyte (PML) homodimers within their corresponding nuclear body.

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。我们开发了一种光谱方法来监测活细胞中的荧光共振能量转移（FRET）。该方法使用双光子激发荧光成像和光谱（TPIS-FRET）在亚细胞成像体积内提供了良好的GFP突变信号光谱分离。与目前基于fret的方法相比，TPIS-FRET不依赖于光滤光片的选择，比例图像分析，或漏校正算法。固有的光学切片能力的TPIS-FRET，蛋白质-蛋白质的相互作用可以识别在离散的亚细胞结构域。为了说明这种技术对检测同型二聚体形成的适用性，我们证明了早幼粒细胞（PML）同型二聚体在其相应核体内的体内关联。