High-resolution gel filtration of the ecdysteroid receptor-DNA complex--an alternative to the electrophoretic mobility shift assay

The mobility shift assay is a well established method for proving binding of protein to DNA. However, this method depends on the stability of the protein-DNA complex during the electrophoretic process. Ecdysteroid receptor shows a strong tendency to aggregate under low-salt conditions of electrophor...

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Bibliographic Details
Published inJournal of chromatography Vol. 587; no. 1; p. 11
Main Authors Ozyhar, A, Kiltz, H H
Format Journal Article
LanguageEnglish
Published Netherlands 29.11.1991
Subjects
Animals
Base Sequence
Binding, Competitive
Chromatography, Gel
DNA - metabolism
DNA-Binding Proteins - metabolism
Drosophila melanogaster
Electrophoresis
Invertebrate Hormones - metabolism
Molecular Sequence Data
Molecular Weight
Oligonucleotides - genetics
Receptors, Steroid - metabolism
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Summary:The mobility shift assay is a well established method for proving binding of protein to DNA. However, this method depends on the stability of the protein-DNA complex during the electrophoretic process. Ecdysteroid receptor shows a strong tendency to aggregate under low-salt conditions of electrophoresis to a non DNA-binding form. We have developed a high-resolution gel filtration method which allows the interaction of ecdysteroid receptor with specific DNA sequences to be studied. The method seems to be generally applicable. It does not depend on the availability of a purified protein. Crude preparations could be used to characterize the stoichiometry and the molecular parameters of the complexes formed between DNA and DNA-binding proteins.
DOI:10.1016/0021-9673(91)85192-I