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.
Hallikas
Outi
O
Molecular/Cancer Biology Program, Institute of Biomedicine, University of Helsinki, Finland.
Taipale
Jussi
J
D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D019921:Biotinylation; D016384:Consensus Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D017930:Genes, Reporter; D049410:Luciferases, Renilla / Q000032:analysis; D008163:Luminescent Measurements; D015336:Molecular Probe Techniques; D009841:Oligonucleotides / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000032:analysis; D014157:Transcription Factors / Q000378:metabolism*
D004268:DNA-Binding Proteins; D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D004247:DNA; D049410:Luciferases, Renilla