A sensitive and rapid gel retention assay for nuclear factor I and other DNA-binding proteins in crude nuclear extracts.

AUTOR(ES)

Schneider, R

RESUMO

The paper describes a rapid and sensitive assay for DNA binding proteins which interact with specific and defined binding sites. It exploits the observation that complexes of proteins and small synthetic DNA fragments (40 bp) containing the protein/DNA binding site can enter native polyacrylamide gels and remain stably associated during electrophoresis under non-denaturing conditions. The assay was applied to nuclear factor I, to its identification and purification from porcine liver, to an analysis of its binding site on adenovirus type 5 DNA and to an exploration of other potential binding sites for DNA binding proteins within the inverted terminal repetition of adenovirus DNA. The extreme sensitivity of the assay which surpasses that of conventional footprint assays by at least two orders of magnitude permitted the identification of nuclear factor I-like activities in Saccharomyces cerevisiae.

Documentos Relacionados

• Rapid isolation of specific DNA-binding proteins and their DNA-binding domains.
• Enrichment of DNA-binding proteins from crude tissue for electrophoretic mobility shift assay using magnetic phospho cellulose particles.
• A rapid microscale procedure for the simultaneous preparation of cytoplasmic RNA, nuclear DNA binding proteins and enzymatically active luciferase extracts.
• A novel DNA-binding motif in the nuclear matrix attachment DNA-binding protein SATB1.
• Exonuclease III protection assay with FRET probe for detecting DNA-binding proteins