Time-resolved luminescence resonance energy transfer imaging of protein-protein interactions in living cells.
Methods Enzymol
; 505: 329-45, 2012.
Article
en En
| MEDLINE
| ID: mdl-22289461
ABSTRACT
Lanthanide-based or luminescence resonance energy transfer (LRET) microscopy can be used to sensitively image interactions between reporter-labeled proteins in living mammalian cells. With LRET, luminescent lanthanide complexes are used as donors, conventional fluorophores are used as acceptors, and donor-sensitized acceptor emission occurs at time scales that reflect the long (~ms) lanthanide emission lifetime. These long-lived signals can be separated from short-lifetime (~ns) sample autofluorescence and directly excited acceptor fluorescence by using pulsed light to excite the specimen and by implementing a short delay (>100 ns) before detection, thereby increasing measurement sensitivity. As practical implementation of time-resolved LRET microscopy requires several potentially unfamiliar experimental techniques, we explicitly describe herein methods to label proteins in living mammalian cells with luminescent terbium complexes, image interactions between terbium-labeled proteins and green fluorescent protein fusions, and quantitatively analyze LRET images.
Texto completo:
1
Base de datos:
MEDLINE
Asunto principal:
Transferencia Resonante de Energía de Fluorescencia
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Proteínas Fluorescentes Verdes
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Luminiscencia
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Rastreo Celular
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Microscopía Fluorescente
Idioma:
En
Revista:
Methods Enzymol
Año:
2012
Tipo del documento:
Article