In Vitro Determination of Temperature-Dependent DNA Binding of the Evening Complex Using Electrophoretic Mobility Shift Assays.
Methods Mol Biol
; 2795: 135-147, 2024.
Article
em En
| MEDLINE
| ID: mdl-38594535
ABSTRACT
Electrophoretic mobility shift assays (EMSAs) of DNA-binding proteins and labeled DNA allow the qualitative and quantitative characterization of protein-DNA complex formation using native (nondenaturing) polyacrylamide or agarose gel electrophoresis. By varying the incubation temperature of the protein-DNA binding reaction and maintaining this temperature during electrophoresis, temperature-dependent protein-DNA interactions can be investigated. Here, we provide examples of the binding of a transcriptional repressor complex called the Evening Complex, comprising the DNA-binding protein LUX ARRYTHMO (LUX), the scaffold protein EARLY FLOWERING 3 (ELF3), and the adapter protein ELF4, to its cognate DNA and demonstrate direct detection and visualization of thermoresponsive binding in vitro. As negative controls we use the LUX DNA-binding domain and LUX full length protein, which do not exhibit temperature-dependent DNA binding.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
DNA
/
Proteínas de Ligação a DNA
Idioma:
En
Revista:
Methods Mol Biol
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2024
Tipo de documento:
Article
País de afiliação:
França