UNIT 12.2 Mobility Shift DNA-Binding Assay Using Gel Electrophoresis
Published Online: 1 MAY 2001
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
Current Protocols in Molecular Biology
How to Cite
Buratowski, S. and Chodosh, L. A. 2001. Mobility Shift DNA-Binding Assay Using Gel Electrophoresis. Current Protocols in Molecular Biology. 36:12.2:12.2.1–12.2.11.
- Published Online: 1 MAY 2001
- Published Print: OCT 1996
The 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.