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Identification of phage antibodies toward the Werner protein by selection on Western blots
Author(s) -
Ravn Peter,
Kjær Svend,
Jensen Kristian Hobolt,
Wind Troels,
Jensen Kim Bak,
Kristensen Peter,
Brosh Robert M.,
Orren David K.,
Bohr Vilhelm A.,
Clark Brian F. C.
Publication year - 2000
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/(sici)1522-2683(20000201)21:3<509::aid-elps509>3.0.co;2-5
Subject(s) - panning (audio) , blot , phage display , antibody , microbiology and biotechnology , biology , polyacrylamide gel electrophoresis , gel electrophoresis , southern blot , nitrocellulose , chemistry , biochemistry , dna , gene , membrane , genetics , paleontology , zoom , enzyme , lens (geology)
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 1.9 × 10 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.