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Updated: Jun 27, 2026

Detecting the Ligand-binding Domain Dimerization Activity of Estrogen Receptor Alpha Using the Mammalian Two-Hybrid Assay
Published on: December 19, 2018
[Establishment of estrogen receptor alpha trans-activation system]
Jie-Ping Li1, Zhi-Hong Xiong, Zhi-Hong Yang
1Beijing Institue of Biotechnology, Beijing 100850, China.
Objective:
To construct estrogen receptor alpha (ERalpha) trans-activation system.
Methods:
The full length ERalpha and its different function regions [(transcriptional activation function 1 (AF1), DNA binding domain (DBD), and transcriptional activation function 2 (AF2)] were amplified from pcDNA3-ERalpha by PCR and cloned into the pGAL vector. The expressions of the recombinant plasmids constructed were detected via immunoblotting. The 293T cells transfected with recombinant plasmids of full length ERalpha, its different function regions and empty vector were divided into 5 groups; each group was divided into 2 parts which were treated with or without estrogen (E(2)). The transcriptional activity of each group was detected in 293T cells after the recombinant plasmid was co-transfected with 0.2 microg of estrogen receptor element luciferase (ERE-LUC) and 0.1 microg of plasmid expressing beta-galactosidase and treated with or without 10 nmol/L E(2) for 24 hours.
Results:
The full length ERalpha and its different function regions were expressed in the 293T cells. Compared with the empty pGAL vector, the transcription activities of full length ERalpha, AF1, AF2 and DBD recombinant plasmids were raised about 20.44 +/- 1.01, 2.09 +/- 0.11, 8.09 +/- 0.30 and 1.05 +/- 0.09 fold, respectively, with the induction of E(2) after transfection in the 293T cells.
Conclusion:
The trans-activation system of ERalpha has been successfully established.
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