Production and characterization of monoclonal antibodies to estrogen-related receptor alpha (ERR伪) and use in immunoaffinity chromatography
- 作者：Amanda M. Esch ; Nancy E. Thompson ; Jennifer A. Lamberski ; Janet E. Mertz ; Richard R. Burgess ; rburgess@wisc.edu ; burgess@oncology.wisc.edu
- 关键词：Estrogen-related receptor alpha ; Nuclear receptor ; Orphan receptor ; Breast cancer ; Cancer ; Immunoaffinity chromatography ; Immunoprecipitation ; Immunofluorescence ; ELISA ; Chromatin immunoprecipitation ; Antibody characterization ; Monoclonal ; Polyol-responsi
- 刊名：Protein Expression and Purification
- 出版年：2012
- 期刊代码：184_10465928
- 类别：bio
- 出版时间：July, 2012
- 卷：84
- 期：1
- 页码：47-58
- 文件大小：1521 K
摘要
Estrogen-related receptor alpha (ERR伪) is an orphan nuclear receptor whose elevated expression is thought to contribute to breast, colon, and ovarian cancers. In order to investigate the role of ERR伪 in human disease, there is a need for immunological reagents suitable for detection and purification of ERR伪. We expressed recombinant human ERR伪 in Escherichia coli, purified the protein, and used it to generate monoclonal antibodies (mAbs) to ERR伪. Nine high-affinity mAbs were chosen for their abilities to detect overexpressed ERR伪 in enzyme-linked immunosorbent assays (ELISAs) and Western blots, after which isotyping and preliminary epitope mapping was performed. The mAbs were all IgG subtypes and reacted with several different regions of full-length ERR伪. A majority of the mAbs were found to be useful for immunoprecipitation of ERR伪, and several could detect DNA-bound ERR伪 in electrophoretic mobility supershift assays (EMSAs) and chromatin immunoprecipitation (ChIP). The suitability of mAbs to detect ERR伪 in immunofluorescence assays was assessed. One mAb in particular, 2ERR10, could specifically detect endogenous ERR伪 in mammary carcinoma cells. Finally, we performed assays to screen for mAbs that gently release ERR伪 in the presence of a low-molecular-weight polyhydroxylated compound (polyol) and nonchaotropic salt. Using gentle immunoaffinity chromatography, we were able to isolate ERR伪 from mammalian cells by eluting with a polyol-salt solution. Our characterization studies show that these monoclonal antibodies perform well in a variety of biochemical assays. We anticipate that these novel reagents will prove useful for the detection and purification of ERR伪 in research and clinical applications.