Journal
BIO-PROTOCOL
Volume 10, Issue 23, Pages -Publisher
BIO-PROTOCOL
DOI: 10.21769/BioProtoc.3843
Keywords
DNase I footprinting; KMnO4 footprinting; Gel retardation assays; EMSAs; Protein-DNA interactions
Categories
Funding
- National Institutes of Health (NIH) [F30GM123632]
- Michigan State University DO/PhD Program
- Intramural Research Program of the NIH, National Institute of Diabetes and Digestive and Kidney Diseases
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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.
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