Assay the protein fractions for the sequence-specific DNA-binding activity using a DNA-binding assay (Brenowitz, et al., 1989; Buratowski and Chodosh, 1996; Baldwin et al., 1996). Estimate the purity of the protein fractions by SDS-PAGE (unit 10.1) followed by silver staining to visualize the protein.
If further purification is desired, combine the fractions that contain the activity and, depending on the KCl concentration, either dilute (using buffer Z without KCl) or dialyze (against buffer Z/0.1 M KCl) to 0.1 M KCl. Then combine the protein fraction with nonspecific competitor DNA (the amount and type of which must be determined experimentally as described in Support Protocol 2) and reapply to either fresh or regenerated DNA affinity resin.
In some instances, proteins might still be bound to the affinity resin after the 1 M salt elution step (step 7). Thus, if the desired protein is not detected in either the column fractions or flowthrough, it is possible that the protein is still on the column, and a wash at a higher salt concentration (e.g., 2 M KCl) may be needed to elute the protein from the resin.