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: (a) the central, highly conserved, DNA-binding domain (DBD) of approximately 66 amino acids, and (b) 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 TRb 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).
GST-TRα1 was expressed in E. coli and purified by a combination of affinity and gel filtration chromatography. GST-TR-α1 has been applied in DNA and protein-protein interaction assays.
The purified fusion protein is greater than 95% homogeneous based on SDS-PAGE analysis. 1 unit equals 1 nanogram of purified protein. 20-200ng is sufficient for an in vitro transcription assay and 100 ng is sufficient for a protein-protein interaction assay.
variable in different lots 1x dilution buffer A: 20 mM Tris-Cl (pH 8.0), 20% Glycerol, 100 mM KCl, 1 mM DTT and 0.2 mM EDTA
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