Combining Gel Retardation and Footprinting to Determine Protein-DNA Interactions of Specific and/or Less Stable Complexes

Meng-Lun Hsieh; Alice Boulanger; Leslie G Knipling; Deborah M Hinton

doi:10.21769/BioProtoc.3843

Combining Gel Retardation and Footprinting to Determine Protein-DNA Interactions of Specific and/or Less Stable Complexes

Bio Protoc. 2020 Dec 5;10(23):e3843. doi: 10.21769/BioProtoc.3843.

Authors

Meng-Lun Hsieh^{1

2}, Alice Boulanger¹, Leslie G Knipling¹, Deborah M Hinton¹

Affiliations

¹ Gene Expression and Regulation Section, Laboratory of Cell and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA.
² Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI, USA.

Abstract

DNA footprinting is a classic technique to investigate protein-DNA interactions. However, traditional footprinting protocols can be unsuccessful or difficult to interpret if the binding of the protein to the DNA is weak, the protein has a fast off-rate, or if several different protein-DNA complexes are formed. Our protocol differs from traditional footprinting protocols, because it provides a method to isolate the protein-DNA complex from a native gel after treatment with the footprinting agent, thus removing the bound DNA from the free DNA or other protein-DNA complexes. The DNA is then extracted from the isolated complex before electrophoresis on a sequencing gel to determine the footprinting pattern. This analysis provides a possible solution for those who have been unable to use traditional footprinting methods to determine protein-DNA contacts.

Keywords: DNase I footprinting; EMSAs; Gel retardation assays; KMnO4 footprinting; Protein-DNA interactions.