Site-specific photobiotinylation of immunoglobulins, fragments and light chain dimers.
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.
Pavlinkova
G
G
Department of Microbiology and Immunology, University of Kentucky, Lexington 40436, USA.
Rajagopalan
K
K
Muller
S
S
Chavan
A
A
Sievert
G
G
Lou
D
D
O'Toole
C
C
Haley
B
B
Kohler
H
H
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001386:Azides / Q000737:chemistry; D001710:Biotin / Q000737:chemistry*; D002469:Cell Separation / Q000379:methods; D005434:Flow Cytometry / Q000379:methods; D015483:HIV Antibodies / Q000737:chemistry; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry; D007128:Immunoglobulin Fragments / Q000737:chemistry*; D007147:Immunoglobulin Light Chains / Q000737:chemistry*; D007075:Immunoglobulin M / Q000737:chemistry; D007136:Immunoglobulins / Q000737:chemistry*; D051379:Mice; D010777:Photochemistry; D011489:Protein Denaturation; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D001386:Azides; D015483:HIV Antibodies; D007140:Immunoglobulin Fab Fragments; D007128:Immunoglobulin Fragments; D007147:Immunoglobulin Light Chains; D007075:Immunoglobulin M; D007136:Immunoglobulins; C055966:immunoglobulin Fv; D001710:Biotin