Selective enrichment of monospecific polyclonal antibodies for antibody-based proteomics efforts
2004 (English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1043, 33-40 p.Article in journal (Refereed) Published
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.
Place, publisher, year, edition, pages
2004. Vol. 1043, 33-40 p.
Affinity chromatography; Antibodies; Immobilized metal-ion affinity chromatography; Protein epitope signature tags; Proteomics
IdentifiersURN: urn:nbn:se:kth:diva-5915DOI: 10.1016/j.chroma.2004.06.008ISI: 000222728800006PubMedID: 15317410ScopusID: 2-s2.0-3242702469OAI: oai:DiVA.org:kth-5915DiVA: diva2:10449
QC 20100824. 23rd International Symposium on the Separation of Proteins Peptides and Polynucleotides. Delray Beach, FL. NOV 09-12, 20032006-06-022006-06-022011-10-18Bibliographically approved