Accumulation of FlAsH/Lumio Green in active mitochondria can be reversed by β-mercaptoethanol for specific staining of tetracysteine-tagged proteins
Accumulation of FlAsH/Lumio Green in active mitochondria can be reversed by β-mercaptoethanol for specific staining of tetracysteine-tagged proteins
复制标题
DOI：
10.1007/s00418-005-0136-3
复制
发表时间：
2006-06-01
影响因子：
2.3
通讯作者：
Stuermer, Claudia A. O.
中科院分区：
文献类型：
--
作者：
Langhorst, Matthias F.;Genisyuerek, Selda;Stuermer, Claudia A. O.
用于活细胞成像的小分子标记领域的最新进展有望克服荧光蛋白大小设置的一些限制。我们在HeLa和N2a细胞的活细胞荧光显微镜下检测了四半胱氨酸-二砷标记系统对Reggie-1/Flotillin-2的影响。在两种类型的细胞中，双砷试剂Flash/Lumio Green积聚在活跃的线粒体中，导致线粒体肿胀。这表明砷引起的毒副作用，当这种标记系统用于活细胞成像时，应该考虑到这一点。在标记过程中加入低浓度的含硫试剂并随后进行高强度的硫醇洗涤，可逆转Flash/Lumio Green的线粒体积累。乙二硫醇和β-硫醇都被证明是有效的。因此，我们建立了一种使用β-硫醇作为硫醇结合位点竞争对手的染色方法，从而使四半胱氨酸标记的Reggie-1/Flotillin-2具有适当的信噪比，从而可以避免更有毒和不便的乙二硫醇。此外，我们还表明，通过串联引入第二个四半胱氨酸序列，染色效率大大提高。
Recent advances in the field of small molecule labels for live cell imaging promise to overcome some of the limitations set by the size of fluorescent proteins. We tested the tetracysteine-biarsenical labeling system in live cell fluorescence microscopy of reggie-1/flotillin-2 in HeLa and N2a cells. In both cell types, the biarsenical staining reagent FlAsH/Lumio Green accumulated in active mitochondria and led to mitochondrial swelling. This is indicative of toxic side effects caused by arsenic, which should be considered when this labeling system is to be used in live cell imaging. Mitochondrial accumulation of FlAsH/Lumio Green was reversed by addition of low concentrations of thiol-containing reagents during labeling and a subsequent high stringency thiol wash. Both ethanedithiol and beta-mercaptoethanol proved to be effective. We therefore established a staining protocol using beta-mercaptoethanol as thiol binding site competitor resulting in a specific staining of tetracysteine-tagged reggie-1/flotillin-2 of adequate signal to noise ratio, so that the more toxic and inconvenient ethanedithiol could be avoided. Furthermore, we show that staining efficiency was greatly enhanced by introducing a second tetracysteine sequence in tandem.