Optimization of BRET saturation assays for robust and sensitive cytosolic protein-protein interaction studies.
Sci Rep
; 12(1): 9987, 2022 06 15.
Article
em En
| MEDLINE
| ID: mdl-35705637
ABSTRACT
Bioluminescence resonance energy transfer (BRET) saturation is a method of studying protein-protein interaction (PPI) upon quantification of the dependence of the BRET signal on the acceptor/donor (AD) expression ratio. In this study, using the very bright Nluc/YFP BRET pair acquired respectively with microplate reader and automated confocal microscopy, we significantly improved BRET saturation assay by extending AD expression detection range and normalizing AD expression with a new BRET-free probe. We next found that upon using variable instead of fixed amount of donor molecules co-expressed with increasing acceptor concentrations, BRET saturation assay robustness can be further improved when studying cytosolic protein, although the relative amounts of dimers (BRETmax) and the relative dimer affinity (BRET50) remain similar. Altogether, we show that our method can be applied to many PPI networks, involving the NF-κB pathway, high-affinity nanobody, rabies virus-host interactions, mTOR complex and JAK/STAT signaling. Altogether our approach paves the way for robust PPI validation and characterization in living cells.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Mapas de Interação de Proteínas
/
Medições Luminescentes
Tipo de estudo:
Diagnostic_studies
Idioma:
En
Revista:
Sci Rep
Ano de publicação:
2022
Tipo de documento:
Article