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Coiled-Coil Tag–Probe Labeling Methods for Live-Cell Imaging of Membrane Receptors

2012; Academic Press; Linguagem: Inglês

10.1016/b978-0-12-391857-4.00018-5

1557-7988

Yoshiaki Yano, Kenichi Kawano, Kaoru Omae, Katsumi Matsuzaki,

Click Chemistry and Applications

Tag–probe labeling methods have advantages over conventional fusion with fluorescent proteins in terms of smaller labels, surface specificity, availability of pulse labeling, and ease of multicolor labeling. With this method, the gene of the target protein is fused with a short tag sequence, expressed in cells, and the protein is labeled with exogenous fluorescent probes that specifically bind to the tag. Various labeling principles, such as protein–ligand interaction, peptide–peptide interaction, peptide–metal interaction, and enzymatic reactions, have been applied to the tag–probe labeling of membrane receptors. We describe our coiled-coil tag–probe method in detail, including the design and synthesis of the tag and probe, labeling procedures, and observations by confocal microscopy. Applications to the analysis of receptor internalization and oligomerization are also introduced.