Rapid development of genetically encoded FRET reporters.
ACS Chem Biol
; 6(7): 685-91, 2011 Jul 15.
Article
em En
| MEDLINE
| ID: mdl-21506563
ABSTRACT
To meet the demand on genetically encoded reporter molecules for live cell imaging, we introduce a new facile combined cloning and FRET reporter analysis strategy. The versatile and fully orthogonal cloning approach involves a set of up to 36 vectors featuring a variety of fluorescent protein FRET pairs and different length linkers. The construct set was successfully applied to two calmodulin-binding proteins, the death-associated protein kinase 1 (DAPK1) and calcium/calmodulin-dependent protein kinase II α (Camk2a). Clone analysis and reporter validation was performed by printing plasmid DNA arrays and subsequent semiautomated microscopy of reversely transfected cells. Characterization of the best performing DAPK1 and Camk2a reporters revealed significant differences in translating calcium signals into kinase responses despite the close functional and structural similarity.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Proteínas de Ligação a Calmodulina
/
Proteínas Recombinantes
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Genes Reporter
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Proteínas Quinases Dependentes de Cálcio-Calmodulina
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Transferência Ressonante de Energia de Fluorescência
/
Proteínas Reguladoras de Apoptose
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Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina
Limite:
Animals
/
Humans
Idioma:
En
Revista:
ACS Chem Biol
Ano de publicação:
2011
Tipo de documento:
Article