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SNAP‐tag, A Multipurpose Visualization Tool for Studying Protein Dynamics in Living Cells
Author(s) -
Sun Luo,
Zhang Aihua,
Ghosh Inca,
Xu MingQun
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.lb207
Subject(s) - internalization , green fluorescent protein , microbiology and biotechnology , live cell imaging , bimolecular fluorescence complementation , intracellular , fluorescence , biochemistry , biology , chemistry , cell , gene , physics , quantum mechanics
The creation of cell lines expressing proteins fused to intrinsically fluorescent tags (e.g., GFP) and their use in cell based studies of protein localization have become routine. The fluorescent signal from such tags cannot be routinely switched on at will, precluding some time‐resolved studies of expression, localization and degradation. A complementary and more flexible imaging system based on a family of small, self‐labeling protein tags is being introduced by New England Biolabs. SNAP‐tag and CLIP‐tag, orthogonal tags with a variety of fluorescent substrates, are suitable for imaging intracellular and cell surface proteins in live cells. These tags can also be used for time‐resolved dual labeling, highly efficient covalent pull‐downs and other biochemical assays. Furthermore, a collection of non‐fluorescent substrates that block SNAP‐ and CLIP‐tag reactivity enable pulse‐chase studies and an examination of the temporal dynamics of nascent protein synthesis and complex formation in live cells. In this report, several studies of protein localization, turnover and receptor internalization using SNAP‐ and CLIP‐tags in living cells are demonstrated. Using two fluorescent dyes in pulse‐chase experiments, we examined the turnover of several intracellular proteins including the Cytochrome c oxidase subunit 8A (COX8A) and the Transferrin Receptor. We demonstrate that fluorescent protein labeling using SNAP‐tag and CLIP‐tag provides a powerful tool to study protein localization, turnover and internalization.