KTH Royal Institute of Technology, Science for Life Laboratory, School of Biotechnology, Stockholm, Sweden.
Nat Methods. 2013 Apr;10(4):315-23. doi: 10.1038/nmeth.2377. Epub 2013 Feb 24.
Imaging techniques such as immunofluorescence (IF) and the expression of fluorescent protein (FP) fusions are widely used to investigate the subcellular distribution of proteins. Here we report a systematic analysis of >500 human proteins comparing the localizations obtained in live versus fixed cells using FPs and IF, respectively. We identify systematic discrepancies between IF and FPs as well as between FP tagging at the N and C termini. The analysis shows that for 80% of the proteins, IF and FPs yield the same subcellular distribution, and the locations of 250 previously unlocalized proteins were determined by the overlap between the two methods. Approximately 60% of proteins localize to multiple organelles for both methods, indicating a complex subcellular protein organization. These results show that both IF and FP tagging are reliable techniques and demonstrate the usefulness of an integrative approach for a complete investigation of the subcellular human proteome.
成像技术，如免疫荧光（IF）和荧光蛋白（FP）融合表达，广泛用于研究蛋白质的亚细胞分布。在这里，我们报告了一项对>500 个人类蛋白质的系统分析，分别比较了使用 FP 和 IF 在活细胞和固定细胞中获得的定位。我们发现 IF 和 FP 之间以及 FP 在 N 和 C 末端标记之间存在系统差异。该分析表明，对于 80%的蛋白质，IF 和 FP 产生相同的亚细胞分布，并且通过两种方法的重叠确定了 250 种以前未定位的蛋白质的位置。大约 60%的蛋白质对于两种方法都定位于多个细胞器，表明蛋白质在亚细胞中有复杂的组织。这些结果表明，IF 和 FP 标记都是可靠的技术，并证明了综合方法对于完整研究人类亚细胞蛋白质组的有用性。
