Combination of Native and Denaturing PAGE for the Detection of Protein Binding Regions in Long Fragments of Genomic DNA

In traditional electrophoresis mobility shift assay (EMSA) a single ³² P-labeled double-stranded DNA oligonucleotide or a restriction fragment bound to a protein is separated from the unbound DNA by polyacrylamide gel electrophoresis (PAGE) under nondenaturing conditions. An extension of this method uses a population of DNA restriction fragments derived from long genomic regions for the identification of fragments containing protein binding regions. Although the method allows simultaneous analysis of large fragments, it is relatively laborious and can be used to detect only fragments containing high affinity protein binding sites. Here we describe an alternative and straightforward strategy which is based on a combination of native and denaturing PAGE. With this strategy restriction fragments, derived from genomic DNA (<10 kb), containing high as well as low affinity protein binding regions may be easily identified.