Combining Gel Retardation And Footprinting To Determine Protein-Dna Interactions Of Specific And/Or Less Stable Complexes
BIO-PROTOCOL(2020)
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.
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Key words
DNase I footprinting, KMnO4 footprinting, Gel retardation assays, EMSAs, Protein-DNA interactions
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