DNA Binding (Gel Retardation Assay) Analysis for Identification of Aryl Hydrocarbon (Ah) Receptor Agonists and Antagonists

Abstract

The gel retardation assay (GRA), also referred as the electromobility shift assay (EMSA), is commonly used technique to examine DNA binding of transcription factors including activated nuclear receptors to their specific DNA recognition sites. GRA of the aryl hydrocarbon receptor (AhR) relies on the in vitro ability of the cytosolic AhR protein complex to convert into its high affinity DNA binding form following its interaction with and activation by an AhR agonist and, as such, the GRA can be used for detection of such ligands. In addition, examination of the ability of a chemical to block agonist-dependent DNA binding of the AhR provides an avenue to identify AhR antagonists. Accordingly, this assay allows relatively rapid screening and identification of both AhR agonists and/or antagonists and unlike cell-based or in vivo assays, it essentially eliminates the confounding effect of cellular metabolism of the test compounds. The GRA can also be used with nuclear extracts obtained from treated cells to further identify and/or characterize compounds capable of stimulating nuclear translocation and DNA binding of the AhR in intact cells. The methods described here can be applied to cytosolic, nuclear and/or whole cell extracts from various species and tissues.