Gel-Shift Analysis and Identification of RXREs and RAREs by PCR-Based Selection

The electrophoretic mobility-shift assay is one of an array of techniques that are used to identify and characterize protein-DNA interactions. Thts method is based on the retardation of a labeled-DNA fragment in a nondenaturmg gel by bound proteins (1 ). The binding specificity and affinity can easily be determined by competition analysis with an excess of specific or nonspecific DNA. The retinoid receptors can interact with their target DNA either in a heterodimeric (retinoic acid receptoketinoic X receptor [RAR/RXR]) or in a homodimeric (RXR/RXR) configuration (2 ). If the protein source is a complex-cellular extract, one can distmguish between these possibilities by comcubation with specific antibodies to the receptors. When antibodies are added to the binding reaction a DNA/receptor/antibody complex will be formed that is further retarded (“supershift”). Alternatively, the antibodies may impair the receptors ability to bind the DNA or to dimerize, resulting in the disappearance of the shifted band. The mobiltty-shift assay can easily be acquired, is fast and very sensitive.