Analysis of DNA-Binding Activity in Neuronal Tissue with the Electrophoretic Mobility-Shift Assay

Abstract

DNA is bound by many proteins in a sequence specific manner. Electrophoretic mobility-shift assays (EMSAs), also known as gel shift assays, are designed to determine the amount and identity of proteins from a particular sample that bind to a specific DNA sequence, usually an enhancer element. The defined DNA sequence is presented as a radiolabeled, synthetic oligonucleotide to a protein extract, which contains DNA-binding proteins. These proteins can then bind to the oligonucleotide. The sample is electrophoresed through a nondenaturing acrylamide gel in a vertical electrophoresis system; the gel is dried and exposed to X-ray film. Whereas the oligonucleotide will move quickly through the gel, oligonucleotides that are bound by protein will move slower and thus be shifted. The more DNA-binding proteins bind to oligonucleotides, the stronger the radioactive signal of the shifted band.