Shift-Western Blotting: Separate Analysis of Protein and DNA from Protein-DNA Complexes.
Methods Mol Biol
; 1312: 355-73, 2015.
Article
en En
| MEDLINE
| ID: mdl-26044017
ABSTRACT
The electrophoretic mobility shift assay (EMSA) is the most frequently used experiment for studying protein-DNA interactions and to identify DNA-binding proteins. Protein-DNA complexes formed during EMSA experiments can be further analyzed by shift-western blotting, where the protein and DNA components contained in a polyacrylamide gel are transferred to stacked membranes First a nitrocellulose membrane retains the proteins while double-stranded DNA passes through the nitrocellulose membrane and binds only to a charged membrane placed below. Immobilized proteins can then be stained with specific antibodies while the DNA can be detected by a radioactive label or a nonradioactive detection system. Shift-western blotting can overcome many limitations of supershift experiments and allows for the analysis of complex protein-DNA complexes containing multiple protein factors. Moreover, proteins and/or DNA may be recovered from membranes after the blotting step for further analysis by other means.
Texto completo:
1
Banco de datos:
MEDLINE
Asunto principal:
ADN
/
Western Blotting
/
Ensayo de Cambio de Movilidad Electroforética
/
Proteínas de Unión al ADN
Tipo de estudio:
Prognostic_studies
Límite:
Animals
/
Humans
Idioma:
En
Revista:
Methods Mol Biol
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
2015
Tipo del documento:
Article