1. Selective enrichment of monospecific polyclonal antibodies for antibody-based proteomics efforts.
- Author
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Agaton C, Falk R, Höidén Guthenberg I, Göstring L, Uhlén M, and Hober S
- Subjects
- Antibodies isolation & purification, Antibody Specificity, Blotting, Western, Antibodies chemistry, Proteomics
- 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.
- Published
- 2004
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