One-step split GFP staining for sensitive protein detection and localization in mammalian cells.
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.
Kaddoum
Lara
L
CNRS, Institute of Pharmacology and Structural Biology (IPBS), Toulouse, France.
Magdeleine
Eddy
E
Waldo
Geoffrey S
GS
Joly
Etienne
E
Cabantous
Stéphanie
S
D000818:Animals; D045744:Cell Line, Tumor; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D042541:Intracellular Space / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins