Abstract
Since its introduction in 1975 the methodology of Kohler and Milstein (1) for production of monoclonal antibody (MAb) from hybridoma cells has been widely used to provide antibodies with a defined specificity. One characteristic feature of this technology is that impure antigens can be used to produce monospecific antibodies that can be utilized to study the functional domains of protein molecules. In this chapter, the use of limited vs complete proteolytic digestion experiments to define the epitope on the antigen recognized by a given MAb is outlined. We describe our studies (2) with the transducin (Gt) α-subunit in which proteolytic digestion, SDS-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, and Edman degradation were used to determine the sequence of the fragments recognized by the MAb 4A. Therefore, reaction times and reagents presented in this chapter may require some modification when a different protein antigen is under investigation.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Kohler, G. and Milstein, C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495–497.
Mazzoni, M. R., Malinski, J. A., and Hamm, H. E. (1991) Structural analysis of rod GTP-binding protein, Gt. J. Biol. Chem. 266, 14,072–14,081.
Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.
Towbin, H., Staehelin, T., and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350–4354.
Matsudaira, P. (1989) Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J. Biol. Chem. 262, 10,035–10,038.
Timmons, T. M. and Dunbar, B. S. (1990) Protein blotting and immunodetection. Methods Enzymol. 182, 679–688.
Matsudaira, P. (1990) Limited N-terminal sequence analysis. Methods Enzymol. 182, 602–613.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1996 Humana Press Inc.
About this protocol
Cite this protocol
Mazzoni, M.R., Artemyev, N.O., Hamm, H.E. (1996). Proteolytic Fragmentation for Epitope Mapping. In: Morris, G.E. (eds) Epitope Mapping Protocols. Methods in Molecular Biology™, vol 66. Humana Press. https://doi.org/10.1385/0-89603-375-9:109
Download citation
DOI: https://doi.org/10.1385/0-89603-375-9:109
Publisher Name: Humana Press
Print ISBN: 978-0-89603-375-7
Online ISBN: 978-1-59259-552-5
eBook Packages: Springer Protocols