کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
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2020590 | 1069193 | 2012 | 12 صفحه PDF | دانلود رایگان |
Estrogen-related receptor alpha (ERRα) 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 ERRα in human disease, there is a need for immunological reagents suitable for detection and purification of ERRα. We expressed recombinant human ERRα in Escherichia coli, purified the protein, and used it to generate monoclonal antibodies (mAbs) to ERRα. Nine high-affinity mAbs were chosen for their abilities to detect overexpressed ERRα 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 ERRα. A majority of the mAbs were found to be useful for immunoprecipitation of ERRα, and several could detect DNA-bound ERRα in electrophoretic mobility supershift assays (EMSAs) and chromatin immunoprecipitation (ChIP). The suitability of mAbs to detect ERRα in immunofluorescence assays was assessed. One mAb in particular, 2ERR10, could specifically detect endogenous ERRα in mammary carcinoma cells. Finally, we performed assays to screen for mAbs that gently release ERRα in the presence of a low-molecular-weight polyhydroxylated compound (polyol) and nonchaotropic salt. Using gentle immunoaffinity chromatography, we were able to isolate ERRα 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 ERRα in research and clinical applications.
► We generated a panel of high-affinity monoclonal antibodies (mAbs) to ERRα.
► Nine mAbs detected ERRα in ELISA and Western blot; mAbs isotyped and epitope-mapped.
► mAbs characterized for use in IP, EMSA, ChIP, and immunofluorescence assays.
► Endogenous ERRα purified in a single step by gentle immunoaffinity chromatography.
► These mAbs are useful for detection and purification of endogenous ERRα.
Journal: Protein Expression and Purification - Volume 84, Issue 1, July 2012, Pages 47–58