Selectivity of antibodies to estrogen receptors alpha and beta (ER alpha and ER beta) for detecting DNA-bound ER alpha and ER beta in vitro

Antibodies are widely used to detect estrogen receptor (ER) in ER-DNA compl exes in electrophoretic mobility shift assays (EMSA). We compared the speci ficity of antibodies raised to different regions of ER alpha or ER beta for detecting recombinant human ER alpha (rhER alpha) and recombinant rat ER b eta (rrER beta) when bound to a consensus estrogen response element (ERE). ER alpha-specific antibodies specifically slowed the migration of the ER-ER E complex by 32 to 84% and inhibited rhER alpha-ERE binding by 17 to 75%. N one of antibodies to ER beta supershifted rhER alpha-ERE complex. Some ER a lpha-specific antibodies increased whereas some decreased rrER beta-ERE bin ding. Anti-ER beta antibodies supershifted different amounts of the rrER be ta-ERE complex. Our results indicate that supershift and inhibition of ER-E RE interaction with a specific antibody are equally reliable in the detecti on of rhER alpha and n-ER beta. ER alpha antibody Ab10, antisera G20 and AT 3B, and ER beta-antiserum Y19 offered the best discrimination between ER al pha and ER beta. Comparison of the peptide sequences against which various antibodies were raised indicate directions for new ER alpha and ER beta- sp ecific antibody development. We conclude that a cognate ER antibody that re tards the migration of the ER-ERE complex by at least 40% or inhibits ER-ER E interaction by at least 8% provides a reliable detection of a specific ER isoform in EMSA. (C) 2000 Elsevier Science Inc. All rights reserved.