Figure 1. Filter binding and gel mobility shift assays. For illustrative purposes, a hypothetical set of interactions between a binding protein (P) and a DNA fragment containing two separate and independent binding sites (x and y) is shown. Separation of a mixture of unbound (UNB) DNA and each of the three possible bound forms, by (a) filter binding and (b) gel mobility shift, is depicted. Note that filter binding does not discriminate between the different bound forms, but the gel mobility shift is able to fractionate the doubly-bound complexes away from the singly-bound species (in this case it is assumed that the two distinct singly-bound species cannot be distinguished solely on the basis of gel mobility).

Figure 2. DNAase I footprinting: an example of the protection class of footprinting assays. Unbound and protein-bound DNA fragments (labelled (asterisk) at one end on one strand) are treated with limiting amounts of DNAase I (no more than one cleavage per molecule). The digestion reactions are fractionated on a denaturing polyacrylamide gel. Protein bound to DNA protects areas from DNAase I attack. The absence of bands corresponding to digestion products generated by cleavage in these areas defines the protein-binding site (the ‘footprint’).

Figure 3. Footprinting interference assay. End-labelled DNA (asterisk) is subjected to limited chemical modification before exposure to the binding protein. The binding reaction is fractionated via a gel mobility shift assay to separate bound (B) from unbound (UNB) species. The DNA in the bound (retarded) band will either be unmodified or contain modifications at positions not affecting binding. The DNA in the unbound band will be comprised of molecules in which modification has occurred at positions affecting interaction with the protein. The DNA from both bands is isolated, subjected to strand cleavage at positions adjacent to the site of modification, and fractionated on a denaturing polyacrylamide gel. Positions which, when modified, affect protein binding will produce corresponding cleavage products only in the sample from the unbound species.