Analysis of estrogen receptor β interacting proteins using pull-down assay and MALDI-MS methods.

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.