[38] Protein-blotting procedures to evaluate interactions of steroid receptors with DNA

Publisher Summary This chapter describes the protein-blotting procedures for evaluating the interactions of steroids receptors with DNA. Selective binding of steroid hormone receptors to hypoxia response element (HRE)-containing DNA fragments has been demonstrated by a variety of techniques for the study of DNA–protein interaction, including DNA–cellulose competitive binding, nitrocellulose filter binding, gel retardation, sucrose gradient shift, DNA footprinting (nuclease protection), and methylation interference assays, in addition to Southwestern blotting. Each of these techniques has its own set of advantages and limitations. Southwestern blotting, unlike nuclease protection or methylation interference studies, does not require the prior purification of the DNA-binding protein and can be used to study DNA–protein interactions in crude cellular extracts or in partially purified protein preparations at any stage of purification. Southwestern blotting offers further advantages in that once a protein extract has been electrophoretically separated and finally immobilized on a nitrocellulose filter, proteins of interest can potentially be characterized by a number of criteria, including relative mobility on the sodium dodecyl sulfate (SDS)-polyacrylamide gel, isoelectric point on two-dimensional gels, DNA-binding activity, ligand binding, or immunoreactivity, if antibodies for the protein are available.