Yeast two-hybrid liquid screening.
Methods Mol Biol
; 1163: 97-107, 2014.
Article
em En
| MEDLINE
| ID: mdl-24841301
ABSTRACT
Yeast two-hybrid (YTH) method consists of a genetic trap that selects for "prey" cDNA products within a library that interact with a "bait" protein of interest. Here, we provide a protocol for YTH screening using a liquid medium screening method, which improves the sensitivity of this technique and streamlines the laborious classic screening in solid medium plates. The method uses a simple series of dilutions with established yeast strains transformed with diverse baits and complex cDNA libraries. This allows for prompt detection of positive clones revealed by liquid growth, due to activation of HIS3 reporter gene. Activation of a second reporter gene and reconstruction of the YTH interaction is highly reproducible using this system. This approach can either be performed using culture flasks or deep-well 96-well plates and the number of interactions obtained is similar, when compared to the classic method. In addition, the liquid screening method is faster and more economical for YTH screening and has the added benefit of automation if 96-well plates are used.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Técnicas do Sistema de Duplo-Híbrido
/
Mapeamento de Interação de Proteínas
/
Proteínas de Ligação a DNA
Tipo de estudo:
Diagnostic_studies
/
Screening_studies
Idioma:
En
Ano de publicação:
2014
Tipo de documento:
Article