Antibody against nuclear thyroid hormone receptors

Area of Science:

• Molecular Biology
• Endocrinology
• Protein Chemistry

Background:

• Thyroid hormone receptors (TRs) are crucial nuclear proteins regulating gene expression.
• Understanding TR structure and function is vital for comprehending thyroid hormone action.
• Specific antibodies are essential tools for studying TR interactions and domains.

Purpose of the Study:

• To purify the rat liver nuclear thyroid hormone receptor.
• To generate a specific antibody against the purified thyroid hormone receptor.
• To characterize the antibody's binding specificity.

Main Methods:

• Multi-step protein purification including hydroxylapatite, gel filtration, and ion-exchange chromatography.
• Immunization of mice with purified receptor to produce polyclonal antibodies.
• Glycerol density gradient sedimentation and immunoprecipitation assays to analyze receptor-antibody interactions.

Main Results:

• Rat liver nuclear thyroid hormone receptor purified to high specific activity (700-1600 pmol T3 binding/mg protein).
• Generated mouse antiserum shifted the sedimentation coefficient of the [125I]T3-receptor complex from 3.4 S to ~7 S.
• Antibody showed reduced immunoprecipitation of a trypsin-digested fragment lacking DNA binding activity, indicating epitope specificity.

Conclusions:

• Successfully produced and characterized an antibody against the rat liver nuclear thyroid hormone receptor.
• The antibody recognizes epitopes distinct from the hormone and core histone binding domains.
• This antibody is a valuable tool for further investigating thyroid hormone receptor structure and function.