Rainbow Trout Glucocorticoid Receptor Overexpression inEscherichia coli:Production of Antibodies for Western Blotting and Immunohistochemistry☆

摘要: Fragments of cDNA that encode the N-terminal and DNA-binding domains (DBD) rainbow trout glucocorticoid receptor (rtGR) were expressed inEscherichia colias fusion proteins with glutathione-S-transferase (GST). The induced by IPTG could readily be detected as 45- 40-kDa bands, respectively, in crude extracts, well purified on glutathione-agarose. These hybrid used to immunize rabbits. antisera produced tested for specificity Western blot analysis using extracts from COS-1 cells transfected an rtGR expression vector liver cells. raised against DBD domain did not detect any bands blots, even at low antiserum dilution. However, protein specifically bound GRE-containing DNA fragments gel-shift assays, retarded complexes supershifted these antibodies. consistently two 104 100 kDa cell allowed specific immunohistochemical staining fish brain pituitary. For first time fish, antibodies will allow GR different cortisol target tissues.