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Ligand‐Assisted Dual‐Site Click Labeling of EGFR on Living Cells
Author(s) -
Yang Yi,
Lin Shixian,
Lin Wei,
Chen Peng R.
Publication year - 2014
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201400057
Subject(s) - click chemistry , förster resonance energy transfer , epidermal growth factor receptor , chemistry , ligand (biochemistry) , azide , biophysics , cycloaddition , membrane , receptor , fluorescence lifetime imaging microscopy , combinatorial chemistry , fluorescence , biochemistry , biology , catalysis , physics , organic chemistry , quantum mechanics
We have developed a dual‐site click labeling strategy for the simultaneous installation of a FRET donor–acceptor pair onto the extracellular domains of epidermal growth factor receptor (EGFR) on living cells. Our method integrates the genetic code expansion strategy, enzyme‐mediated protein labeling, and ligand‐assisted Cu I ‐catalyzed azide–alkyne cycloaddition (CuAAC) into a tri‐step labeling procedure. This enabled cis ‐membrane FRET imaging of EGFR under living conditions. This procedure might be generally applicable for dual‐site labeling and cis ‐membrane FRET analysis of the domain–domain dynamics of important mammalian cell‐surface receptors.