Unit

UNIT 6.8 Mobility Shift DNA-Binding Assay Using Gel Electrophoresis

  1. Stephen Buratowski1,
  2. Lewis A. Chodosh2

Published Online: 1 AUG 2001

DOI: 10.1002/0471141755.ph0608s13

Current Protocols in Pharmacology

Current Protocols in Pharmacology

How to Cite

Buratowski, S. and Chodosh, L. A. 2001. Mobility Shift DNA-Binding Assay Using Gel Electrophoresis. Current Protocols in Pharmacology. 13:6.8:6.8.1–6.8.12.

Author Information

  1. 1

    Harvard Medical School, Boston, Massachusetts

  2. 2

    University of Pennsylvania, Philadelphia, Pennsylvania

Publication History

  1. Published Online: 1 AUG 2001
  2. Published Print: JUN 2001

Abstract

DNA-binding assay using nondenaturing polyacrylamide gel electrophoresis (PAGE) provides a simple, rapid, and extremely sensitive method for detecting sequence-specific DNA-binding proteins. Proteins that bind specifically to an end-labeled DNA fragment retard the mobility of the fragment during electrophoresis, resulting in discrete bands corresponding to the individual protein-DNA complexes. The assay described in this unit can be used to test binding of purified proteins or of uncharacterized factors found in crude extracts. This assay also permits quantitative determination of the affinity, abundance, association rate constants, dissociation rate constants, and binding specificity of DNA-binding proteins. Three additional protocols describe a competition assay using unlabeled competitor DNA, an antibody supershift assay, and multicomponent gel shift assays.