Shevtsova Z, Malik J M I, Michel U, Schöll U, Bähr M, Kügler S
Department of Neurology; University of Göttingen, Medical School, Waldweg 33, 37073 Göttingen, Germany.
Eur J Neurosci. 2006 Apr;23(8):1961-9. doi: 10.1111/j.1460-9568.2006.04725.x.
Functional characterization of disease-related proteins, their splice variants and dominant negative mutants in the context of complex CNS tissues such as brain and retina is frequently assessed by in vivo gene transfer. For correct interpretation of results it is imperative that the protein under investigation is unambiguously detected in the transduced cell types and can be distinguished from any endogenously expressed physiological variants. Therefore the first systematic evaluation of epitope tags used to trace ectopically expressed proteins in the central nervous system is presented here. Substantial differences in the performances of various epitope tag-antibody combinations with respect to sensitivity, specificity and influence of the epitope tag on the fusion protein are elucidated. Epitope tags already established for protein detection in vitro and to some extent in vivo (c-Myc, HA and FLAG tags) were immunohistochemically detected with high sensitivity. However, detection of these tags revealed problems with background staining and we also document structural and functional influence of the tags on the fusion protein. In order to prevent such unwanted side-effects, epitope tags which have not yet been used for in vivo applications (IRS, EE and AU1 tags) were characterized in brain, retina and cultured neurons. While use of the IRS and EE tags was hindered by low sensitivity or specificity, optimal results were obtained with the AU1 epitope, which may develop into a standard tool for detection of ectopic protein expression in the central nervous system.
在诸如脑和视网膜等复杂中枢神经系统组织的背景下，疾病相关蛋白、其剪接变体和显性负性突变体的功能特性常通过体内基因转移来评估。为了正确解释结果，至关重要的是在所转导的细胞类型中能明确检测到所研究的蛋白，并且能将其与任何内源性表达的生理变体区分开来。因此，本文首次对用于追踪中枢神经系统中异位表达蛋白的表位标签进行了系统评估。阐明了各种表位标签 - 抗体组合在灵敏度、特异性以及表位标签对融合蛋白的影响方面的显著差异。已在体外以及在一定程度上在体内用于蛋白检测的表位标签（c - Myc、HA和FLAG标签）通过免疫组织化学检测具有高灵敏度。然而，这些标签的检测显示出背景染色问题，并且我们还记录了标签对融合蛋白的结构和功能影响。为了防止此类不良副作用，对尚未用于体内应用的表位标签（IRS、EE和AU1标签）在脑、视网膜和培养的神经元中进行了特性分析。虽然IRS和EE标签的使用因灵敏度或特异性低而受到阻碍，但AU1表位获得了最佳结果，它可能会发展成为检测中枢神经系统中异位蛋白表达的标准工具。
