Nature Methods2, 521 - 527 (2005)
Published online: 22 June 2005; | doi:10.1038/nmeth771
Enzymatic detection of protein translocation
Tom S Wehrman1, Clayton L Casipit1, 2, Nevin M Gewertz1
& Helen M Blau1
1
Baxter Laboratory for Genetic Pharmacology, Departments of Microbiology and Immunology, and Molecular Pharmacology, 269 Campus Drive, CCSR 4225A, Stanford University School of Medicine, Stanford, California 94305, USA.
2
KAI Pharmaceuticals, Inc., 270 Littlefield Avenue, South San Francisco, California 94080, USA.
Fundamental to eukaryotic cell signaling is the regulation of protein function by directed localization. Detection of these events has been largely qualitative owing to the limitations of existing technologies. Here we describe a method for quantitatively assessing protein translocation using proximity-induced enzyme complementation. The complementation assay for protein translocation (CAPT) is derived from -galactosidase and comprises one enzyme fragment, , which is localized to a particular subcellular region, and a small complementing peptide, , which is fused to the protein of interest. The concentration of in the immediate vicinity of correlates with the amount of enzyme activity obtained in a dose- and time-dependent manner, thus acting as a genetically encoded biosensor for local protein concentration. Using CAPT, inducible protein movement from the cytosol to the nucleus or plasma membrane was quantitatively monitored in multiwell format and in live mammalian cells by flow cytometry.
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-galactosidase and comprises one enzyme fragment, 
, which is localized to a particular subcellular region, and a small complementing peptide, 
, which is fused to the protein of interest. The concentration of 
