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J Chromatogr A. 2004 Jul 16;1043(1):33-40.

Selective enrichment of monospecific polyclonal antibodies for antibody-based proteomics efforts.

Author information

  • 1Department of Biotechnology, Royal Institute of Technology, AlbaNova University Center, KTH, Stockholm, Sweden.

Abstract

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.

PMID:
15317410
[PubMed - indexed for MEDLINE]
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