Immunolabeling artifacts and the need for live-cell imaging
Immunolabeling artifacts and the need for live-cell imaging
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DOI：
10.1038/nmeth.1855
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发表时间：
2012-02-01
期刊：
影响因子：
48
通讯作者：
Giepmans, Ben N. G.
中科院分区：
文献类型：
--
作者：
Schnell, Ulrike;Dijk, Freark;Giepmans, Ben N. G.
荧光融合蛋白彻底改变了活细胞中蛋白质的检测。尽管如此，使用这些蛋白质的研究受到了批评，因为与免疫荧光研究相反，蛋白质被修饰和异位表达。然而，在细胞内引入免疫试剂会导致蛋白质提取或重新定位，而不能反映体内情况。在这里，我们讨论了免疫荧光标记的陷阱，往往很少受到关注，并认为在死亡，透化细胞的免疫染色实验应补充活细胞成像时，仔细检查蛋白质定位。
Fluorescent fusion proteins have revolutionized examination of proteins in living cells. Still, studies using these proteins are met with criticism because proteins are modified and ectopically expressed, in contrast to immunofluorescence studies. However, introducing immunoreagents inside cells can cause protein extraction or relocalization, not reflecting the in vivo situation. Here we discuss pitfalls of immunofluorescence labeling that often receive little attention and argue that immunostaining experiments in dead, permeabilized cells should be complemented with live-cell imaging when scrutinizing protein localization.