Format

Send to

Choose Destination
ACS Chem Biol. 2011 May 20;6(5):401-6. doi: 10.1021/cb100431e. Epub 2011 Jan 20.

Chromophore-assisted light inactivation of HaloTag fusion proteins labeled with eosin in living cells.

Abstract

Chromophore-assisted light inactivation (CALI) is a potentially powerful tool for the acute disruption of a target protein inside living cells with high spatiotemporal resolution. This technology, however, has not been widely utilized, mainly because of the lack of an efficient chromophore as the photosensitizing agent for singlet oxygen ((1)O(2)) generation and the difficulty of covalently labeling the target protein with the chromophore. Here we choose eosin as the photosensitizing chromophore showing 11-fold more production of ((1)O(2)) than fluorescein and about 5-fold efficiency in CALI of β-galactosidase by using an eosin-labeled anti-β-galactosidase antibody compared with the fluorescein-labeled one. To covalently label target protein with eosin, we synthesize a membrane-permeable eosin ligand for HaloTag technology, demonstrating easy labeling and efficient inactivation of HaloTag-fused PKC-γ and aurora B in living cells. These antibody- and HaloTag-based CALI techniques using eosin promise effective biomolecule inactivation that is applicable to many cell biological assays in living cells.

PMID:
21226520
DOI:
10.1021/cb100431e
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for American Chemical Society
Loading ...
Support Center