ER protein was detected on Western blots and by immunohistochemistry using the H222 rat anti-human ER monoclonal antibody (H222 mAb; gift of Dr. G.L. Greene, University of Chicago). The precise epitope recognized by the H222 mAb is not known. Though preliminary, current evidence indicates that this antibody is specific for ERa (Dr. G.L. Greene, personal communication). Specificity of the H222 mAb was demonstrated by Western blot analysis of total protein extracted from porcine uteri (0, 15, and 120 days of age) according to the procedure of Glatstein and Yeh, exactly as described by Goyal et al. with the following modifications. buy ampicillin
Extracted protein (50 |xg per lane) was subjected to SDS-PAGE, using a 10% separating gel and the Protean II Electrophoresis Cell system (Bio-Rad, Richmond, CA). The separated proteins were transferred to nitrocellulose membrane according to procedures recommended for the Bio-Rad Trans-Blot Electrophoretic Transfer Cell. To visualize transferred proteins, the membrane was stained with 0.1% (w:v) Ponceau S in 1% acetic acid so that individual lanes could be identified and cut into strips. Strips were destained in Tween 20 (0.5% w:v)/Tris-buffered saline and probed with H222. Controls included substitution of an irrelevant rat IgG (Jackson Immunological Research Laboratories, West Grove, PA) for H222 mAb and elimination of the primary antibody. Protein bands were visualized using the ECL che-miluminescence system (Amersham Life Science, Cleveland, OH) and Kodak X-OMAT LS film (Eastman Kodak, Rochester, NY).