Purification and Quality Control The His-tag recombinant protein is purified by affinity chromatography in combination with FPLC columns. The purified Thyroid Hormone Receptor alpha 1 is greater than 95% homogeneous based on SDS-PAGE analysis.
Unit Definition (Activity) 1 unit equals 1 nanogram of purified protein. 20 ng is sufficient for an in vitro transcription assay and 100 ng is sufficient for a protein-protein interaction assay.
Applications TR-α1 has been applied in in vitro transcription assays, DNA and protein-protein interactions assays.
Formulation and Storage The protein is in 20mM Tris-HCl pH7.9,100mM NaCl, 0.2mM EDTA, 1mM DTT and 20% glycerol. Stored at -70°C before use. Avoid repeated freeze thaw cycles.
Background Nuclear receptors form the largest known family of transcription factors and have a crucial role in nearly all aspects of vertebrate development and adult physiology by transducing the effects of hormones into transcriptional responses (1). The family is defined by two domains: (α) the central, highly conserved, DNA-binding domain (DBD) of approximately 66 amino acids, and (β) the C-terminal, structurally conserved, ligand-binding domain (LBD) of approximately 250 amino acids (2, 3). The amino-terminal regions are least conserved among nuclear receptor sequences. This domain is highly divergent between TRα and TRβ isoforms, which suggests differential roles in transcriptional regulation. In addition, alternative splicing of the TRβ gene generates two isoforms, TRβ1 and TRβ2 with completely different amino-terminal domains (4). Unliganded TR inhibits the formation of a functional pre-initiation complex, through direct interaction with TBP and transcription factor IIB (5-7). In addition, in the absence of ligand TR has been shown to repress transcription through recruitment of a corepressor complex, which also includes Sin3A and histone deacetylase (8, 9). Ligand binding releases the corepressor complex and recruits a coactivator complex that includes multiple histone acetyltransferases, including a steroid receptor family coactivator, p300/CREB-binding protein–associated factor (PCAF), and CREB binding protein (CBP) (10-14).
References 1. Mangelsdorf, D. J., et al., (1995) Cell 83, 835–839 2. Glass, C. K. (1994) Endocrinol. Rev. 15, 391–407 3. Moras, D., et al., (1998) Curr. Opin. Cell Biol. 10, 384-391 4. Lazar M.A. (1993) Endocr. Rev. 14, 184–193 5. Fondell J.D. et al., (1993) Genes Dev. 7, 1400–1410 6. Fondell J.D., et al., (1996) Mol. Cell Biol. 16, 281–287 7. Baniahmad A., et al., (1993) Proc. Natl. Acad. Sci. USA 90, 8832–8836 8. Zhang X.J.M., et al., (1996) J. Biol. Chem. 271, 14825–14833 9. Heinzel, T., et al., (1997) Nature 387, 43-48 10. Onate, S. A., et al., (1995) Science 270, 1354-1357 11. Kamei, Y., et al., (1996) Cell 85, 403-414 12. Blanco, J.C., et al., (1998) Genes Dev. 12, 1638-1651 13. Fondell, J.D., et al., (1996) Proc. Natl. Acad. Sci. USA 93, 8329-8333 14. Wolffe, A., et al., (1997) Genes Cells 2, 291-302
DISCLAIMER
This products is recommended For RESEARCH USE ONLY and is Not qualified for Use in Diagnostic or Therapeutic Procedures.
