Selectivity of antibodies to estrogen receptors alpha and beta (ERalpha and ERbeta) for detecting DNA-bound ERalpha and ERbeta in vitro.
Antibodies are widely used to detect estrogen receptor (ER) in ER-DNA complexes in electrophoretic mobility shift assays (EMSA). We compared the specificity of antibodies raised to different regions of ERalpha or ERbeta for detecting recombinant human ERalpha (rhERalpha) and recombinant rat ERbeta (rrERbeta) when bound to a consensus estrogen response element (ERE). ERalpha-specific antibodies specifically slowed the migration of the ER-ERE complex by 32 to 84% and inhibited rhERalpha-ERE binding by 17 to 75%. None of antibodies to ERbeta supershifted rhERalpha-ERE complex. Some ERalpha-specific antibodies increased whereas some decreased rrERbeta-ERE binding. Anti-ERbeta antibodies supershifted different amounts of the rrERbeta-ERE complex. Our results indicate that supershift and inhibition of ER-ERE interaction with a specific antibody are equally reliable in the detection of rhERalpha and rrERbeta. ERalpha antibody Ab10, antisera G20 and AT3B, and ERbeta-antiserum Y19 offered the best discrimination between ERalpha and ERbeta. Comparison of the peptide sequences against which various antibodies were raised indicate directions for new ERalpha and ERbeta- specific antibody development. We conclude that a cognate ER antibody that retards the migration of the ER-ERE complex by at least 40% or inhibits ER-ERE interaction by at least 8% provides a reliable detection of a specific ER isoform in EMSA.
Tyulmenkov
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VV
Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine, KY 40292, USA.
Klinge
C M
CM
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D000918:Antibody Specificity / Q000276:immunology*; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000276:immunology*; D004586:Electrophoresis; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D047628:Estrogen Receptor alpha; D047629:Estrogen Receptor beta; D006801:Humans; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D011960:Receptors, Estrogen / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D018168:Receptors, Retinoic Acid / Q000276:immunology; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology; D020218:Response Elements
D000906:Antibodies; D004268:DNA-Binding Proteins; D000939:Epitopes; D047628:Estrogen Receptor alpha; D047629:Estrogen Receptor beta; D011960:Receptors, Estrogen; D018168:Receptors, Retinoic Acid; D011994:Recombinant Proteins; D004247:DNA