Abstract
To examine protein-DNA binding, a series of assays such as electrophoretic mobility shift assays (EMSA) have been developed. However, these are time consuming experiments, and contain several difficulties including the use of radioisotopes or specific antibodies. In addition, during gel electrophoresis, subtle binding may be dissociated. To overcome such difficulties, we developed a novel in vitro DNA-protein binding assay. This assay is based on a fluorescence-labeled DNAs and GST-fused proteins. After washing steps, immunological reaction was performed on the GST beads to detect binding activities that are measured by luminometer in solution in seconds. This technique is convenient because of its simple steps, in which radioisotopes are not required, and a small amount of garbage is produced. Thus, it may be useful for the screening of toxic compounds. By using this technique, we first confirmed the interaction of nuclear receptor (NR) with nuclear hormone response element (NRE). Moreover, we showed that GST-coactivator- or -corepressor-NR-NRE interactions. These results were compatible to those using EMSA or transient transfection-based reporter gene assays. Furthermore, we can apply this technique to study protein-protein or protein-RNA interactions.In summary, we developed a novel in vitro DNA-protein binding assay and this technique is useful to screen a large amount of DNA-protein interactions in a short time. [J Physiol Sci. 2007;57 Suppl:S74]
