Animal Science Abstract
The porcine glucocorticoid receptor: sequencing a 2.1kb cDNA fragment and raising specific polyclonal antibodies for western blotting, immunoprecipitation and immunohistochemistry
M. Gutscher1, S. Eder2, M. Müller2 and R. Claus1,A
ACorresponding author e-mail: thsekret@uni-hohenheim.de
1Fachgebiet Tierhaltung und Leistungsphysiologie, Institut für Tierhaltung und Tierzüchtung (470a), Universität Hohenheim, Garbenstr. 17, 70599 Stuttgart, Germany2Institut für Tierzucht und Genetik, Veterinärmedizinische Universität Wien, Veterinärplatz 1, 1210 Vienna, Austria
Abstract
Glucocorticoids and their tissue receptors are involved in many metabolic and developmental processes. Until now only two short fragments with a total length of 200 amino acids were known from the glucocorticoid receptor of the pig. Therefore we sequenced the main part (2.1kb) of the porcine receptor. In addition, we subcloned a cDNA fragment of this sequence coding for 135 aa of the modulatory region in a pET expression vector. The protein fragment was expressed in E. coli as a his-tag fusion protein. In the SDS-PAGE, the crude E. coli extracts showed an enrichment of a 15kDa protein which corresponds to the estimated molecular weight for the receptor fragment.
After lysis and Ni-NTA affinity chromatography under denaturing conditions the protein was further purified either by dialysis (native protein) or by SDS-PAGE (linearized form). Both forms were emulsified together in adjuvant and used for rabbit immunization.
The resulting antibodies were characterized by western blot analysis, immunoprecipitation, and additionally by immunohistochemistry. Western blot analysis confirmed the binding of the denatured protein by the antiserum and revealed a high binding affinity. Immunoprecipitation demonstrated that both the occupied and unoccupied forms of the receptor are detected. The specificity of the antiserum for pGCR was additionally demonstrated by immunohistochemistry.
Keywords: amino acid sequences, glucocorticoids, immunohistochemistry, pigs, western blotting
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