Evaluation of Fluorophores to Label SNAP-Tag Fused Proteins for Multicolor Single-Molecule Tracking Microscopy in Live Cells
Evaluation of Fluorophores to Label SNAP-Tag Fused Proteins for Multicolor Single-Molecule Tracking Microscopy in Live Cells
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DOI：
10.1016/j.bpj.2014.06.040
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发表时间：
2014-08-19
影响因子：
3.4
通讯作者：
Subramaniam, Vinod
中科院分区：
文献类型：
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作者：
Bosch, Peter J.;Correa, Ivan R., Jr.;Subramaniam, Vinod
单分子跟踪技术已成为研究蛋白质动力学及其在细胞复杂环境中的组织的广泛使用的技术。特别是，膜受体的时空分布是一个活跃的研究领域，由于其在信号转导的调节中的假定作用。SNAP标签是一种固有的单价和高度特异性的遗传标签，用于将荧光标记附着到感兴趣的蛋白质上。目前很少有信息可供选择的最佳荧光染料的单分子显微镜利用SNAP标签标记系统。我们调查了6个绿色和16个红色的可激发染料在活细胞中SNAP标签融合蛋白的单分子显微镜下的适用性。我们确定了非特异性结合水平和光稳定性的这些染料共轭物结合时，SNAP标签融合的膜蛋白在活细胞中。我们发现，只有有限的子集的染料测试是适合单分子跟踪显微镜。结果表明，仔细选择的染料共轭SNAP-底物标记SNAP标签融合蛋白是非常重要的，因为许多染料遭受快速光漂白或高度非特异性染色。这些特征似乎是不可预测的，这促使需要进行系统的调查在这里提出。我们已经开发了一种用于评估最佳染料的方案，对于我们评估的条件，我们发现Dy 549和CF 640是单分子追踪测试的最佳选择。使用一个最佳的染料对，我们也证明了双色单分子成像SNAP标签融合蛋白的可能性。这项调查提供了一系列SNAP标记的荧光底物的生物物理和成像特性的概述，使选择最佳的染料和条件的SNAP标记的融合蛋白在真核细胞系中的单分子成像。
Single-molecule tracking has become a widely used technique for studying protein dynamics and their organization in the complex environment of the cell. In particular, the spatiotemporal distribution of membrane receptors is an active field of study due to its putative role in the regulation of signal transduction. The SNAP-tag is an intrinsically monovalent and highly specific genetic tag for attaching a fluorescent label to a protein of interest. Little information is currently available on the choice of optimal fluorescent dyes for single-molecule microscopy utilizing the SNAP-tag labeling system. We surveyed 6 green and 16 red excitable dyes for their suitability in single-molecule microscopy of SNAP-tag fusion proteins in live cells. We determined the nonspecific binding levels and photostability of these dye conjugates when bound to a SNAP-tag fused membrane protein in live cells. We found that only a limited subset of the dyes tested is suitable for single-molecule tracking microscopy. The results show that a careful choice of the dye to conjugate to the SNAP-substrate to label SNAP-tag fusion proteins is very important, as many dyes suffer from either rapid photobleaching or high nonspecific staining. These characteristics appear to be unpredictable, which motivated the need to perform the systematic survey presented here. We have developed a protocol for evaluating the best dyes, and for the conditions that we evaluated, we find that Dy 549 and CF 640 are the best choices tested for single-molecule tracking. Using an optimal dye pair, we also demonstrate the possibility of dual-color single-molecule imaging of SNAP-tag fusion proteins. This survey provides an overview of the photophysical and imaging properties of a range of SNAP-tag fluorescent substrates, enabling the selection of optimal dyes and conditions for single-molecule imaging of SNAP-tagged fusion proteins in eukaryotic cell lines.