Selective enrichment of monospecific polyclonal antibodies for antibody-based proteomics efforts.
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.
Department of Biotechnology, Royal Institute of Technology, AlbaNova University Center, KTH, Stockholm, Sweden.
Charlotta
C
Agaton
Ronny
R
Falk
Ingmarie
I
Höidén Guthenberg
Lovisa
L
Göstring
Mathias
M
Uhlén
Sophia
S
Hober
D000906:Antibodies / Q000737:chemistry* / Q000302:isolation & purification; D000918:Antibody Specificity; D015153:Blotting, Western; D040901:Proteomics
D000906:Antibodies