Monitoring of in vitro and in vivo translation of green fluorescent protein and its fusion proteins by fluorescence correlation spectroscopy.
Cytometry
; 44(1): 1-6, 2001 May 01.
Article
en En
| MEDLINE
| ID: mdl-11309802
BACKGROUND: Because the process of protein translation is an event of sparse molecules, the measurement requires high sensitivity. One of the candidates for studying the molecules is fluorescence correlation spectroscopy (FCS), which gleans quantitative information from fluctuating fluorescence signals in a diluted solution. METHODS: Using FCS, the translation products of expression plasmid for green fluorescent protein (GFP) and its fusion proteins were measured in vitro and in vivo. RESULTS: In in vitro translation, the number of products increased linearly for 90 min upon concentration of the plasmid. The autocorrelation function for GFP was fitted with a one-component model with a diffusion time of 0.18 ms, which was identical to the value expected from the molecular weight. In the cases of GFP- tagged hypoxia-inducible factor-1 alpha and glucocorticoid receptor, each fitting result was significantly improved with a two-component model. The slow component with a diffusion time of 6 ms appeared to be related to the ribosome or polysome. In response to the addition of dexamethasone, the nuclear translocation from cytosol clearly induced the decrease in number of molecules in the focal point. CONCLUSIONS: FCS permits monitoring of the number of molecules translated in vitro and in vivo, the translation rate, and the molecular weight.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Factores de Transcripción
/
Biosíntesis de Proteínas
/
Proteínas Nucleares
/
Genes Reporteros
/
Hormona de Crecimiento Humana
/
Proteínas de Unión al ADN
/
Proteínas Luminiscentes
Tipo de estudio:
Prognostic_studies
Límite:
Animals
/
Humans
Idioma:
En
Revista:
Cytometry
Año:
2001
Tipo del documento:
Article
País de afiliación:
Japón
Pais de publicación:
Estados Unidos