Multiexcitation Fluorogenic Labeling of Surface, Intracellular, and Total Protein Pools in Living Cells.
Multiexcitation Fluorogenic Labeling of Surface, Intracellular, and Total Protein Pools in Living Cells.
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DOI：
10.1021/acs.bioconjchem.6b00169
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发表时间：
2016-06-15
影响因子：
4.7
通讯作者：
Bruchez MP
中科院分区：
文献类型：
--
作者：
Naganbabu M;Perkins LA;Wang Y;Kurish J;Schmidt BF;Bruchez MP
孔雀石绿色（MG）是一种荧光染料，其在与其工程化同源蛋白（荧光团活化蛋白（FAP））结合时显示荧光增强。能量转移供体，如花青和罗丹明染料已与MG共轭，以改变荧光复合物的光谱性质，其中供体染料通过Förster共振能量转移将能量转移到MG复合物，导致在远红外区域的结合条件荧光发射。在这篇文章中，我们使用一个紫色激发染料作为供体敏化的MG-FAP复合物的远红光发射。将两种蓝色发射荧光香豆素染料偶联到MG上，并评价通过其次级激发带向MG-FAP复合物的能量转移。6，8-二氟-7-羟基香豆素-3-羧酸（Pacific blue，PB）在紫外光（405 nm）激发下，在远红外区表现出最有效的能量转移和最大亮度。这些蓝-红（BluR）串联染料在光谱上与其他串联染料不同，并且能够产生具有大斯托克斯位移（>250 nm）的MG-FAP复合物的荧光图像。这些染料是细胞可渗透的，用于标记细胞内蛋白质。与标记细胞外蛋白的细胞不可渗透的hexa-Cy 3-MG（HCM）染料一起使用，我们能够使用与不同染料组合以影响不同光谱特性的一个荧光标记来可视化细胞外、细胞内和总细胞蛋白池。
Malachite green (MG) is a fluorogenic dye that shows fluorescence enhancement upon binding to its engineered cognate protein, a fluorogen activating protein (FAP). Energy transfer donors such as cyanine and rhodamine dyes have been conjugated with MG to modify the spectral properties of the fluorescent complexes, where the donor dyes transfer energy through Förster resonance energy transfer to the MG complex resulting in binding-conditional fluorescence emission in the far-red region. In this article, we use a violet-excitable dye as a donor to sensitize the far-red emission of the MG-FAP complex. Two blue emitting fluorescent coumarin dyes were coupled to MG and evaluated for energy transfer to the MG-FAP complex via its secondary excitation band. 6,8-Difluoro-7-hydroxycoumarin-3-carboxylic acid (Pacific blue, PB) showed the most efficient energy transfer and maximum brightness in the far-red region upon violet (405 nm) excitation. These blue-red (BluR) tandem dyes are spectrally varied from other tandem dyes and are able to produce fluorescence images of the MG-FAP complex with a large Stokes shift (>250 nm). These dyes are cell-permeable and are used to label intracellular proteins. Used together with a cell-impermeable hexa-Cy3-MG (HCM) dye that labels extracellular proteins, we are able to visualize extracellular, intracellular, and total pools of cellular protein using one fluorogenic tag that combines with distinct dyes to effect different spectral characteristics.