Agaton Charlotta, Falk Ronny, Höidén Guthenberg Ingmarie, Göstring Lovisa, Uhlén Mathias, Hober Sophia
Department of Biotechnology, Royal Institute of Technology, AlbaNova University Center, KTH, Stockholm, Sweden.
J Chromatogr A. 2004 Jul 16;1043(1):33-40. doi: 10.1016/j.chroma.2004.06.008.
A high stringency protocol, suitable for systematic purification of polyclonal antibodies, is described. The procedure is designed to allow the generation of target protein-specific antibodies suitable for functional annotation of proteins. Antibodies were generated by immunization with recombinantly produced affinity-tagged target proteins. To obtain stringent recovery of the antibodies, a two-step affinity chromatography principle was devised to first deplete the affinity tag-specific antibodies followed by a second step for affinity capture of the target protein-specific antibodies. An analytical dot-blot array system was developed to analyze the cross-reactivity of the affinity-purified antibodies. The results suggest that the protocol can be used in a highly parallel and automated manner to generate mono-specific polyclonal antibodies for large-scale, antibody-based proteomics efforts, i.e. affinity proteomics.
本文描述了一种适用于多克隆抗体系统纯化的高严谨度方案。该程序旨在生成适用于蛋白质功能注释的靶蛋白特异性抗体。通过用重组生产的亲和标签靶蛋白免疫来产生抗体。为了严格回收抗体，设计了两步亲和层析原理，首先去除亲和标签特异性抗体，然后第二步亲和捕获靶蛋白特异性抗体。开发了一种分析性斑点印迹阵列系统来分析亲和纯化抗体的交叉反应性。结果表明，该方案可高度并行且自动化地用于大规模基于抗体的蛋白质组学研究，即亲和蛋白质组学，以生成单特异性多克隆抗体。
