ISSN:
0173-0835
Keywords:
Phage display antibody
;
Two-dimensional polyacrylamide gel electrophoresis
;
Chemistry
;
Biochemistry and Biotechnology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Chemistry and Pharmacology
Notes:
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 × 103 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.
Additional Material:
1 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/elps.1150191044
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