Determination of dissociation constant of the NFκB p50/p65 heterodimer using fluorescence cross-correlation spectroscopy in the living cell.
Biochem Biophys Res Commun
; 436(3): 430-5, 2013 Jul 05.
Article
em En
| MEDLINE
| ID: mdl-23751347
ABSTRACT
Two-laser-beam fluorescence cross-correlation spectroscopy (FCCS) is promising technique that provides quantitative information about the interactions of biomolecules. The p50/p65 heterodimer is the most abundant and well understood of the NFκB dimers in most cells. However, the quantitative value of affinity, namely the K(d), for the heterodimer in living cells is not known yet. To quantify the heterodimerization of the IPT domain of p50/p65 in the living cell, we used two-laser-beam FCCS. The K(d) values of mCherry2- and EGFP-fused p50 and p65 were determined to be 0.46 µM in the cytoplasm and 1.06 µM in the nucleus of the living cell. These results suggest the different binding affinities of the p50/p65 heterodimer in the cytoplasm and nucleus of the living cell and different complex formation in each region.
Palavras-chave
Dissociation constant; EGFP; FCCS; FCS/FCCS; Fluorescence measurement; Heterodimer; IPT; K(d); LSM; Live cell imaging; NFkB; NFκB; NLS; dissociation constant; enhanced green fluorescent protein; fluorescence cross-correlation spectroscopy; immunoglobulin-like plexin transcription factor; laser scanning microscopy; mCherry tandem dimer; mCherry(2); nuclear factor kappa B; nuclear localization signal; p50/p65
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Espectrometria de Fluorescência
/
Núcleo Celular
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Citoplasma
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Subunidade p50 de NF-kappa B
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Fator de Transcrição RelA
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Multimerização Proteica
Limite:
Humans
Idioma:
En
Revista:
Biochem Biophys Res Commun
Ano de publicação:
2013
Tipo de documento:
Article
País de afiliação:
Japão