CNRS, Institute of Pharmacology and Structural Biology (IPBS), Toulouse, France.
Biotechniques. 2010 Oct;49(4):727-8, 730, 732 passim. doi: 10.2144/000113512.
Although epitope tags are useful to detect intracellular proteins and follow their localization with antibodies, background and nonspecific staining often remain problematic. We describe a simple assay based on the split GFP complementation system. Proteins tagged with the 15-amino acid GFP 11 fragment are detected with a solution of the recombinant nonfluorescent complementary GFP 1-10 fragment to reconstitute a fluorescent GFP. In contrast to antibody-based staining methods, this one-step assay presents high specificity and very low background of fluorescence, thus conferring higher signal-to-noise ratios. We demonstrate that this new application of the split GFP tagging system facilitates detection of proteins displaying various subcellular localizations using flow cytometry and microscopy analysis.
尽管表位标签可用于检测细胞内蛋白质并通过抗体追踪其定位，但背景和非特异性染色仍然是个问题。我们描述了一种基于 GFP 分裂互补系统的简单检测方法。通过与重组非荧光互补 GFP 1-10 片段溶液孵育，带有 GFP11 片段 15 个氨基酸标签的蛋白质被重新组装成荧光 GFP，从而被检测到。与基于抗体的染色方法相比，这种一步法检测具有很高的特异性和非常低的荧光背景，从而提高了信号与噪声的比值。我们证明，GFP 分裂标签系统的这一新应用通过流式细胞术和显微镜分析可方便地检测具有各种亚细胞定位的蛋白质。
