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Updated: Apr 24, 2026
Detecting the Ligand-binding Domain Dimerization Activity of Estrogen Receptor Alpha Using the Mammalian Two-Hybrid Assay
Published on: December 19, 2018
Analysis of estrogen receptor β interacting proteins using pull-down assay and MALDI-MS methods
Mahendra Kumar Thakur1, Vijay Paramanik
1Laboratory of Biochemistry and Molecular Biology, Department of Zoology, Banaras Hindu University, Varanasi, 221005, Uttar Pradesh, India, mkt_bhu@yahoo.com.
Abstract:
Estrogen mediates a plethora of functions through well-characterized estrogen receptor (ER)α and ERβ after recruiting a number of interacting proteins. Various laboratories including ours have focused on the identification of ERβ interacting proteins using different methods including matrix-assisted laser desorptive ionization (MALDI), which is a powerful technique in proteomics to identify new proteins present in low abundance. We have identified ERβ interacting proteins resolved by one-dimensional preparatory sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and two-dimensional SDS-PAGE followed by MALDI-MS. In this chapter, a detailed method of pull-down assay, SDS-PAGE, MALDI-MS, and immunoblotting along with the use of software for identification of interacting proteins have been described. Such methods are useful to identify the interacting proteins, which may predict the function and molecular mechanism of action that is helpful for developing therapeutic strategies.
Insights
Researchers identified proteins interacting with estrogen receptor beta (ERβ) using proteomics techniques. These methods help understand ERβ function and develop new therapies.
Area of Science:
- Molecular Biology
- Proteomics
- Endocrinology
Background:
- Estrogen exerts diverse functions via estrogen receptors (ERα and ERβ) by interacting with various proteins.
- Identifying these interacting proteins is crucial for understanding receptor mechanisms and developing therapeutic strategies.
Purpose of the Study:
- To describe detailed methods for identifying estrogen receptor beta (ERβ) interacting proteins.
- To highlight the utility of these methods in predicting protein function and guiding therapeutic development.
Main Methods:
- Utilized pull-down assays, one-dimensional and two-dimensional SDS-PAGE, and MALDI-MS to resolve and identify proteins.
- Employed immunoblotting and specialized software for accurate protein identification.
- Focused on identifying low-abundance proteins interacting with ERβ.
Main Results:
- Successfully identified ERβ interacting proteins using the described proteomic techniques.
- The methods demonstrated effectiveness in resolving and characterizing protein complexes.
Conclusions:
- The described methods provide a robust approach for identifying ERβ interacting proteins.
- Understanding these interactions aids in elucidating molecular mechanisms and developing targeted therapies for estrogen-related conditions.
