Selective enrichment of monospecific polyclonal antibodies for antibody-based proteomics efforts
- 作者：Agaton ; Charlotta ; Falk ; Ronny ; Hö ; idé ; n Guthenberg ; Ingmarie ; Gö ; string ; Lovisa ; et. al.
- 关键词：Proteomics ; Immobilized metal-ion affinity chromatography ; Affinity chromatography ; Antibodies ; Protein epitope signature tags
- 刊名：Journal of Chromatography A
- 出版年：2004
- 期刊代码：47_00219673
- 类别：ch
- 出版时间：July 16, 2004
- 卷：1043
- 期：1
- 页码：33-40
- 文件大小：252 K
摘要
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.