Department of Cell Biology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.
Nat Methods. 2012 Jan 30;9(2):152-8. doi: 10.1038/nmeth.1855.
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.
荧光融合蛋白的出现彻底改变了活细胞内蛋白质的检测方式。不过，这些蛋白质的研究仍然受到质疑，因为与免疫荧光研究相比，这些蛋白质会发生修饰和异位表达。然而，将免疫试剂引入细胞内会导致蛋白质的提取或重定位，无法反映体内情况。本文讨论了免疫荧光标记中常被忽视的一些陷阱，并认为在研究蛋白质定位时，应将固定、通透细胞的免疫染色实验与活细胞成像实验结合起来。
