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Mobility shift DNA-binding assay using gel electrophoresis.

S Buratowski1, L A Chodosh

  • 1Harvard Medical School, Boston, Massachusetts, USA.

Current Protocols in Molecular Biology
|February 12, 2008
PubMed
Summary
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This study introduces a simple, sensitive DNA-binding assay using nondenaturing polyacrylamide gel electrophoresis (PAGE). It effectively detects sequence-specific DNA-binding proteins and quantifies their binding characteristics.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • Detecting sequence-specific DNA-binding proteins is crucial for understanding gene regulation.
  • Existing methods can be complex or lack sensitivity.

Purpose of the Study:

  • To present a simple, rapid, and highly sensitive method for detecting sequence-specific DNA-binding proteins.
  • To enable quantitative analysis of protein-DNA interactions.

Main Methods:

  • Nondenaturing polyacrylamide gel electrophoresis (PAGE) of end-labeled DNA fragments incubated with proteins.
  • Analysis of retarded DNA fragment mobility to identify protein-DNA complexes.

Main Results:

  • The DNA-binding assay successfully detects sequence-specific DNA-binding proteins.

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  • It allows for quantitative determination of binding affinity, abundance, and kinetics.
  • Additional protocols for competition, supershift, and multicomponent assays are described.

    • Conclusions:

    • Nondenaturing PAGE is a versatile and sensitive technique for studying DNA-binding proteins.
    • The assay is applicable to both purified proteins and factors in crude extracts.
    • This method facilitates comprehensive characterization of protein-DNA interactions.