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Optical lock-in detection of FRET using synthetic and genetically encoded optical switches.

ABSTRACT: The Förster resonance energy transfer (FRET) technique is widely used for studying protein interactions within live cells. The effectiveness and sensitivity of determining FRET, however, can be reduced by photobleaching, cross talk, autofluorescence, and unlabeled, endogenous proteins. We present a FRET imaging method using an optical switch probe, Nitrobenzospiropyran (NitroBIPS), which substantially improves the sensitivity of detection to <1% FRET efficiency. Through orthogonal optical control of the colorful merocyanine and colorless spiro states of the NitroBIPS acceptor, donor fluorescence can be measured both in the absence and presence of FRET in the same FRET pair in the same cell. A SNAP-tag approach is used to generate a green fluorescent protein-alkylguaninetransferase fusion protein (GFP-AGT) that is labeled with benzylguanine-NitroBIPS. In vivo imaging studies on this green fluorescent protein-alkylguaninetransferase (GFP-AGT) (NitroBIPS) complex, employing optical lock-in detection of FRET, allow unambiguous resolution of FRET efficiencies below 1%, equivalent to a few percent of donor-tagged proteins in complexes with acceptor-tagged proteins.

SUBMITTER: Mao S

PROVIDER: S-EPMC2480664 | biostudies-literature | 2008 Jun

REPOSITORIES: biostudies-literature

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Optical lock-in detection of FRET using synthetic and genetically encoded optical switches.

Mao Shu S Benninger Richard K P RK Yan Yuling Y Petchprayoon Chutima C Jackson David D Easley Christopher J CJ Piston David W DW Marriott Gerard G

Biophysical journal 20080215 11

The Förster resonance energy transfer (FRET) technique is widely used for studying protein interactions within live cells. The effectiveness and sensitivity of determining FRET, however, can be reduced by photobleaching, cross talk, autofluorescence, and unlabeled, endogenous proteins. We present a FRET imaging method using an optical switch probe, Nitrobenzospiropyran (NitroBIPS), which substantially improves the sensitivity of detection to <1% FRET efficiency. Through orthogonal optical contro ...[more]

PMID: 18281383

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Dataset Information

Optical lock-in detection of FRET using synthetic and genetically encoded optical switches.

Publications

Optical lock-in detection of FRET using synthetic and genetically encoded optical switches.

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