High-throughput assay for determining specificity and affinity of protein-DNA binding interactions

Limited information exists for the binding specificities of many important transcription factors. To address this, we have previously developed a microwell- based assay for directly measuring the affinity of DNA- protein binding interactions. We describe here the detailed protocol for determining sequence specificities of DNA- binding proteins using this assay. The described method is rapid; after preparation of the reagents, the assay can be run in a single day, and its throughput can be increased further by automation. The method is quantitative but requires prior knowledge of one high- affinity binding site for the protein of interest. The protocol can be adapted for determining the effect of protein modifications and protein- protein interactions on DNA- binding specificity, and for engineering proteins with new DNA- binding specificities. In addition, the method is suitable for high- throughput screening to identify proteins or small molecules that modulate protein- DNA binding interactions.