Esch Amanda M, Thompson Nancy E, Lamberski Jennifer A, Mertz Janet E, Burgess Richard R
McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, Wisconsin, USA.
Protein Expr Purif. 2012 Jul;84(1):47-58. doi: 10.1016/j.pep.2012.04.020. Epub 2012 May 4.
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.
雌激素相关受体α（ERRα）是一种孤儿核受体，其表达升高被认为与乳腺癌、结肠癌和卵巢癌有关。为了研究ERRα在人类疾病中的作用，需要适合检测和纯化ERRα的免疫试剂。我们在大肠杆菌中表达重组人ERRα，纯化该蛋白，并用其制备针对ERRα的单克隆抗体（mAb）。选择了9种高亲和力的mAb，因其能够在酶联免疫吸附测定（ELISA）和蛋白质免疫印迹中检测过表达的ERRα，之后进行了亚型鉴定和初步表位作图。这些mAb均为IgG亚型，可与全长ERRα的几个不同区域发生反应。发现大多数mAb可用于ERRα的免疫沉淀，有几种mAb能够在电泳迁移率超迁移分析（EMSA）和染色质免疫沉淀（ChIP）中检测与DNA结合的ERRα。评估了mAb在免疫荧光测定中检测ERRα的适用性。特别是一种mAb，即2ERR10，能够特异性检测乳腺癌细胞中的内源性ERRα。最后，我们进行了检测，以筛选在低分子量多羟基化合物（多元醇）和非离液盐存在下能温和释放ERRα的mAb。使用温和免疫亲和色谱法，我们能够通过用多元醇 - 盐溶液洗脱从哺乳动物细胞中分离出ERRα。我们的表征研究表明，这些单克隆抗体在各种生化检测中表现良好。我们预计这些新型试剂将被证明在研究和临床应用中对ERRα的检测和纯化有用。
