Fluorescent labelling of proteins without compromising their activity is crucial for determining their spatiotemporal localization while retaining their functionality. Spot-tag is a 12-amino acid peptide recognized by a single-domain nanobody. Here we introduce the spot-tag as a labelling strategy for proteins in fixed and living plant cells, using as an example the microtubule motor centromeric protein E-related Kinesin 7.3. Spot-tagging of ectopically introduced Kinesin 7.3 does not interfere with microtubules and spot staining results in a close-grained fluorophore labelling revealing a localization pattern that resembles beads-on-a-string. We anticipate that our protocol will apply to many more demanding protein cellular targets, offsetting activity perturbations and low photon quantum yields imposed by other protein-tagging approaches.

中文翻译：

---

使用斑点标记在植物细胞中进行蛋白质检测和定位

在不影响蛋白质活性的前提下，对蛋白质进行荧光标记对于确定其时空定位至关重要。点标签是被单域纳米抗体识别的12个氨基酸的肽。在这里，我们以微管马达着丝粒蛋白E相关的驱动蛋白7.3为例，介绍斑点标签作为固定和活植物细胞中蛋白质的标记策略。异位引入的Kinesin 7.3的斑点标记不会干扰微管，并且斑点染色会导致荧光团标记紧密，从而揭示出类似于串珠的定位模式。我们预计，我们的方案将适用于更多要求更高的蛋白质细胞靶标，从而抵消了其他蛋白质标记方法带来的活性扰动和低光子量子产率。