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Rapid isolation of phage displayed antibodies to β‐actin eluted from two‐dimensional electrophoresis gel
Author(s) -
Zhou JianNian,
Linder Stig,
Franzén Bo,
Auer Gert,
Hochstrasser Denis F.,
Persson Mats A. A.
Publication year - 1998
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/elps.1150191044
Subject(s) - biotinylation , antibody , phage display , microbiology and biotechnology , gel electrophoresis , biology , chemistry , genetics
We describe a simple and efficient procedure which can be used to prepare antibodies to proteins extracted by two‐dimensional gel electrophoresis (2‐DE), using β‐actin as a model. Protein was electroeluted from a stained gel, biotinylated and used for selection of phage from a semisynthetic phage antibody library. After four rounds of selection using 50 ng β‐actin per cycle, approximately 8 × 10 3 phage were recovered. Antibody fragments were prepared from 21 randomly picked clones. Six of eighteen (6/18) antibody‐positive clones produced antibody fragments reacting against β‐actin in an enzyme linked immunosorbent assay (ELISA). Sequencing of the HC‐CDR3‐region showed that all six clones were independent isolates, suggesting that a large number of independent phage antibody reactivities were generated.