Specific Detection of His-Tagged Proteins with Recombinant Anti-His Tag scFv-Phosphatase or scFv-Phage Fusions
Open Access
- 1 January 1997
- journal article
- Published by Taylor & Francis in BioTechniques
- Vol. 22 (1) , 140-149
- https://doi.org/10.2144/97221rr01
Abstract
Using a cell-bound immunogen, we have generated a monoclonal antibody, 3D5, that recognizes carboxy-terminal oligo-histidine tags (His tags) on a wide variety of proteins. From this monoclonal antibody, we have generated a single-chain fragment of the variable domains (scFv), a dimeric scFv-alkaline phosphatase fusion and an oligovalent scFv-display phage. The antibody in its various formats is an effective tool used in fluorescence-activated cell sorting analysis, the BIAcore® method, Western blots and enzyme-linked immunosorbent assay (ELISA). Western blots and ELISAs can be developed directly by using crude extracts of E. coli cells that produce the scFv-alkaline phosphatase fusion, thus providing an inexhaustable and convenient supply of detection reagent. Alternatively, oligovalent scFv-displaying phage can be used directly from culture supernatants for this purpose. The dissociation constants, KD, of the peptide KGGHHHHH (KD = 4 × 10-7 M) and of imidazole (KD = 4 × 10-4 M) were determined. Molecular...Keywords
This publication has 0 references indexed in Scilit: