Fig. 3.

RIME demonstrates specificity and peptide coverage of ERβ antibodies. Eight ERβ antibodies were assessed by RIME in MDA-MB-231-ERβ+/– cells. Coverage of the protein relates to green areas on the peptide maps, indicating peptides identified by MS with false discovery rate of ≤1% (mean of 2 biological replicates). (A) E2F1 antibody was applied to MDA-MB-231-ERβ– and MDA-MB-231-ERβ+ conditions as a positive control, as E2F1 is a ubiquitously expressed protein. (B) ERβ antibody tests: ‘ERβ ranking’ indicates where ERβ features in a list of proteins purified by the antibody, ranked by number of unique peptides identified in MS, giving an indication of antibody specificity. NCL-ER-BETA failed to identify ERβ. (C) Negative controls: All of the ERβ antibodies were tested in MDA-MB-231-ERβ– cells, to confirm absence of ERβ expression in the non-induced condition. Mouse IgG antibodies were used to identify non-specific peptides pulled down by the IP. None of the IgG antibodies purified ERβ.