Experimental Strategies for the Identification of DNA-Binding Proteins

This article describes approaches for identifying proteins that bind conserved or functional DNA motifs. It discusses the use of consensus sequence databases to identify candidate proteins capable of binding a DNA motif of interest and then explains the potential uses of sophisticated mass spectrometry technology. DNA-binding proteins are most commonly identified by electrophoretic mobility-shift assay (EMSA) or DNase I footprinting. Each of these methods is described, and their advantages and limitations are outlined. It is important to stress that each of the strategies discussed in this article may identify one or more proteins that bind a DNA element of interest. However, none of the strategies will necessarily lead to the protein that is the functionally relevant regulator of the control element in the context of the endogenous locus. Regardless of how a DNA-binding protein is isolated and identified, it merely becomes a candidate regulator of the gene of interest.