Rabbit anti erα polyclonal antibody

Frozen tissues were homogenized in PER-Tissue Protein Extraction Buffer (1:20 w:v) (Pierce, Rockford, IL) containing 100 μM phenylmethylsulfonyl fluoride. The homogenates were centrifuged at 20,000 g for 10 min at 4°C. Supernatants (cytosols) were aliquoted and kept at −80°C freezer until use. Protein concentrations were determined using the bicinchoninic acid (BCA) protein assay. Equal amount of proteins were loaded onto each lane of a 12% polyacrylamide gel. The proteins were separated and electroblotted onto a polyvinyl difluoride (PVDF) membrane (Bio-Rad) at 25 V for 60 min using a Trans-Blot (TE77, GE) semidry transfer apparatus. Membranes were blocked in 5% nonfat dry milk for 1 h at room temperature and then incubated with mouse anti-PCNA polyclonal antibody, rabbit anti-BOULE polyclonal antibody, rabbit anti-ERα polyclonal antibody, rabbit anti-ERβ polyclonal antibody or mouse anti-β-actin polyclonal antibody (1:1000; Santa Cruz Biotechnology, Inc.) overnight at 4°C. After incubation with horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Inc.), immunoblots were visualized on film using SuperSignal Chemiluminescence Substrate (Pierce Biotechnology).

Uterus and vagina were resuspended in lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 5 mM EDTA, 0.1% sodium dodecyl sulfate (SDS), 0.5% NP-40) containing 10 mM phenylmethylsulfonyl fluoride (PMSF) and 2 mg/mL aprotinin. The protein was obtained to detect the levels of ERα and ERβ in target tissue by western blotting. The western blot protocol and semi-quantitative analysis were carried out as described [42 (link)]. The antibody of rabbit anti-ERα polyclonal antibody (dilution 1/200, Santa cruz Biotechnology) or mouse anti-ERβ monoclonal antibody (dilution 1/1,000, Abcam Biotechnology) was used. All experiments were done in triplicate. The relative quantity of each antibody was measured by Alpha Ease FC (Fluorchem FC2) software. The density ratio of protein to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (dilution 1/1,000, Cell Signaling Technology) was calculated from the band density.