Methodologies for Isolating Estrogen-Responsive Proteins as Markers of Environmental Toxicants

New and refined methods for the purification of two major estrogen-responsive proteins in murine uterine luminal fluid (ULF) are described. Lacto-ferrin (LF; approx. 70 kDa), was purified using modifications of procedures previously reported. Using column chromatography and electroelution, the protein was purified to a single band demonstrated by gel electrophoresis. An antibody to the purified protein was then raised in rabbits. Rabbit serum was IgG purified and used in Western blotting and immunocytochemical techniques. The serum IgG purified LF antibody demonstrated only one band in Western blotting procedures using enhanced chemiluminescence (ECL) as a detection method. Another estrogen-inducible protein with an approximate molecular weight of 63 kDa in mouse ULF, purified to a single band by gel electrophoresis and electroelution, was determined to be complement C3 through sequence analysis. Similar to LF, C3 has been previously described as an estrogen-responsive protein in the rodent and human reproductive tract. C3 antibody was purchased commercially and was used for Western blotting and immunohistochemistry. Since interest is increasing in identifying biological markers of hormone-disrupting chemicals, the purification techniques described in this report can be useful in identifying proteins that are potential markers of estrogen action. Antibodies to these proteins can then be used as markers of environmental estrogenic toxicants. In fact, LF and C3 are currently being used to screen for sensitive markers of estrogenicity. Following the methods described in this report, other proteins can be identified and used to screen for possible effects of environmental agents.