Polyclonal Anti-Peptide Antibodies Against Both Activated and Unactivated Forms of the Human Glucocorticoid Receptor
Abstract
The human glucocorticoid receptor (hGR) is a member of a superfamily of ligand-responsive transcription factors. The hGR is comprised of a unique amino-terminus (or hypervariable region), a central DNA-binding domain and a carboxyl terminus (or ligand-binding domain). Regulation of hGR expression is multifactorial, involving transcriptional, post-transcriptional, and post-translational components. In order to study this regulation in detail, we sought to develop specific anti-hGR antibodies. On the basis of hydrophilicity/hydrophobicity ratios of the protein molecule, as well as secondary structure predictions, a peptide corresponding to amino acid residues Cys245-Thr272 within the amino terminus was synthesized and conjugated to keyhole limpet hemacyanin (KLH) anti-hGR antisera were generated in three New Zealand white rabbits. Immunoabsorption of [3H] triamcinolone acetonide ([3H]TA)-labeled hGR showed all three antisera recognized the native form of the protein. In each case, the concentration of antisera required to absorb 50% of the receptor was less than that for polyclonal antisera previously raised against the purified intact receptor protein. Preincubation of the antisera with saturating concentrations of the immunizing peptide completely blocked the immunoabsorption of the (3H]TA-hGR hormone-receptor complex . Incubation of the ligand-receptor complex with the antipeptide antisera caused a discrete increase in the sedimentation coefficient of both activated and unactivated [3H]TA-hGR complexes on sucrose gradients. In addition, a major immunoreactive protein with a molecular weight of 92 kDa, corresponding to the molecular weight of the hGR, was identified by the antisera by Western blot analysis, indicating the antisera also react with the denatured form of the hGR . These combined results demonstrate the usefulness of the antisera in the biochemical characterization of the native and denatured hGR.
Document Details
- Document Type
- Technical Report
- Publication Date
- Apr 30, 1993
- Accession Number
- AD1011296
Entities
People
- Delia C. Tang
Organizations
- Uniformed Services University of the Health Sciences