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.
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References
Thakur MK, Sharma PK (2006) Aging of brain: role of estrogen. Neurochem Res 31: 1389–1398
Thakur MK, Paramanik V (2009) Role of steroid hormone coregulators in health and diseases. Horm Res 71:194–200
Paramanik V, Thakur MK (2012) Estrogen receptor β and its domains interact with casein kinase 2, phosphokinase C, and N-myristoylation sites of mitochondrial and nuclear proteins in mouse brain. J Biol Chem 287:22305–22316
Paramanik V, Thakur MK (2010) Interaction of estrogen receptor associated protein (ERAP) 140 with ER β decreases, but its expression increases in aging mouse cerebral cortex. Cell Mol Neurobiol 30:961–966
Ghosh S, Thakur MK (2009) Interaction of estrogen receptor-alpha ligand binding domain with nuclear proteins of aging mouse brain. J Neurosci Res 15:2591–2600
Barmack NH, Bilderback TR, Li H, Qian Z, Yakhnitsa V (2004) Activity-dependent expression of acyl-coenzyme A-binding protein in retinal muller glial cells evoked by optokinetic stimulation. J Neurosci 24:1023–1033
Vorum H, Hager H, Christensen BM, Nielsen S, Honoré B (1999) Human calumenin localizes to the secretory pathway and is secreted to the medium. Exp Cell Res 248:473–481
Sambrook J, Fritsch EF, Maniatis T (2004) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Ausubel FM, Brent R, Kungstone RE, Moore DD, Seidman JG, Smith JA, Struhl K (1995) Short protocol of molecular biology. Wiley, New York
Hulo N, Bairoch A, Bulliard V, Cerutti L, Cuche BA, de Castro E, Lachaize C, Langerdijk-Genevaux PS, Sinrist CJ (2008) The 20 years of PROSITE. Nucleic Acids Res 36:D245–D249
Acknowledgements
Financial supports from the Indian Council of Medical Research (ICMR) and Department of Biotechnology (BT/PR3593/Med/14/468/2003), Government of India, to M.K.T. are highly acknowledged. The authors declare no conflict of interest.
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Thakur, M.K., Paramanik, V. (2014). Analysis of Estrogen Receptor β Interacting Proteins Using Pull-Down Assay and MALDI-MS Methods. In: Castoria, G., Auricchio, F. (eds) Steroid Receptors. Methods in Molecular Biology, vol 1204. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-1346-6_16
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DOI: https://doi.org/10.1007/978-1-4939-1346-6_16
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Publisher Name: Humana, New York, NY
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