Thakur Mahendra Kumar, Paramanik Vijay
Laboratory of Biochemistry and Molecular Biology, Department of Zoology, Banaras Hindu University, Varanasi, 221005, Uttar Pradesh, India,
Methods Mol Biol. 2014;1204:187-95. doi: 10.1007/978-1-4939-1346-6_16.
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.
雌激素在招募多种相互作用蛋白后，通过特征明确的雌激素受体（ER）α和ERβ介导大量功能。包括我们实验室在内的多个实验室都致力于使用不同方法鉴定ERβ相互作用蛋白，其中包括基质辅助激光解吸电离（MALDI），这是蛋白质组学中用于鉴定低丰度新蛋白的强大技术。我们已经通过一维制备型十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶电泳（PAGE）和二维SDS-PAGE，然后进行MALDI-MS鉴定出了ERβ相互作用蛋白。在本章中，详细描述了下拉分析法、SDS-PAGE、MALDI-MS和免疫印迹法，以及用于鉴定相互作用蛋白的软件的使用方法。这些方法有助于鉴定相互作用蛋白，从而预测其功能和分子作用机制，这对制定治疗策略很有帮助。
