Identification of phage antibodies toward the Werner protein by selection on Western blots.
A procedure was established for selecting phage antibodies (phage-abs) from phage-displayed antibody repertoires by panning against proteins, separated by sodium dodecyl phosphate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto nitrocellulose membranes (Western blots). This immobilization strategy is applicable for secondary rounds of panning in selections against semipurified proteins, and directs the selection toward antibodies suitable as immunochemical reagents in Western blots. In model experiments, enrichment factors as high as 1.9x10(5) were obtained in a single round of panning. Furthermore, we demonstrate the application of this approach by selection of phage-abs recognizing the human Werner protein, which is defective in a premature aging syndrome.
Ravn
P
P
Department of Molecular and Structural Biology, University of Aarhus, Denmark. raven@biobase.dk
Kjaer
S
S
Jensen
K H
KH
Wind
T
T
Jensen
K B
KB
Kristensen
P
P
Brosh
R M
RM
Orren
D K
DK
Bohr
V A
VA
Clark
B F
BF
D000914:Antibodies, Viral / Q000276:immunology*; D001435:Bacteriophages / Q000276:immunology*; D001483:Base Sequence; D015153:Blotting, Western; D016172:DNA Fingerprinting; D004265:DNA Helicases / Q000276:immunology*; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D005090:Exodeoxyribonucleases; D006801:Humans; D016133:Polymerase Chain Reaction; D053484:RecQ Helicases; D000071657:Werner Syndrome Helicase
D000914:Antibodies, Viral; D017931:DNA Primers; D005090:Exodeoxyribonucleases; D004265:DNA Helicases; D053484:RecQ Helicases; C098785:WRN protein, human; D000071657:Werner Syndrome Helicase