Site-specific photobiotinylation of immunoglobulins, fragments and light chain dimers
| Autor: | Boyd E. Haley, Carol O'Toole, Ashok J. Chavan, Krishnan Rajagopalan, Heinz Kohler, Gail Sievert, Dingyuan Lou, Gabriela Pavlinkova, Sybille Müller |
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| Rok vydání: | 1997 |
| Předmět: |
Azides
Protein Denaturation Photochemistry Immunology Biotin Immunoglobulins Cell Separation HIV Antibodies law.invention Immunoglobulin Fab Fragments Mice chemistry.chemical_compound law Ribose Tumor Cells Cultured Animals Humans Immunology and Allergy Nucleotide Immunoglobulin Fragments chemistry.chemical_classification biology Photoaffinity labeling Antibodies Monoclonal Flow Cytometry Molecular biology Immunoglobulin M chemistry Biochemistry Polyclonal antibodies Biotinylation biology.protein Recombinant DNA Immunoglobulin Light Chains Nucleoside |
| Zdroj: | Journal of Immunological Methods. 201:77-88 |
| ISSN: | 0022-1759 |
| DOI: | 10.1016/s0022-1759(96)00214-1 |
| Popis: | Herein we report a new method to rapidly photoinsert biotin into a specific and highly conserved site on the Ig structure using a mild photochemical activation step. This site resides in the Fv fragment and involves invariant residues which provide base stacking interactions to the purine ring of ATP (Rajagopalan et al. (1996) Proc. Natl. Acad. Sci. USA 93, 6019-6024). Biotin was coupled to either the phosphate or the ribose of the 8-azidopurine nucleotide or nucleoside photoaffinity probe and shown to insert into the affinity site efficiently. Several monoclonal and polyclonal antibodies, as well as enzymatic and recombinant antibody fragments and light chain dimers were photoaffinity biotinylated and used in ELISA, FACS and Western blots. The selectivity of this site-specific biotinylation method also allows for biotinylation of antibodies in culture supernatants and immune sera without prior purification. Because the biotinylation takes place under physiological conditions and within a short time period, photobiotinylation would be the preferred method for antibodies which are easily damaged by classical non-site specific random biotinylation chemistry. |
| Databáze: | OpenAIRE |
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