Rapid Epitope Mapping by Carboxypeptidase Digestion and Immunoblotting

  • Philip S. Low
  • Jie Yuan
Part of the Springer Protocols Handbooks book series (SPH)


Conventional methods for locating the epitope of an antibody on an antigen all require amino acid sequencing at some stage of the protocol. The protein footprinting approach, for example, employs arbitrary proteolysis or chemical modification to locate the sequence that is protected by the bound antibody (1,2); however, the protected region still must be sequenced to identify its position on the immunogenic protein. Limited protease digestion of an antigen followed by Western blotting with the desired antibody has also been exploited for identifying crossreactive peptides (3,4), but again the stained peptide must be sequenced to pinpoint its location in the original antigen. Localization of epitopes by comparison of an antibody’s ability to recognize a series of highly homologous proteins has been used to relate differences in specificity with known amino acid substitutions (5,6). However, such families of homologous proteins are rare, and manual generation of homologous families by site-directed mutagenesis can be very time-consuming. Finally, competitive inhibition of antibody binding by synthetic or natural peptides derived from the antigen can often disclose the desired epitope (7,8); nonetheless, the sequence of the competitive peptide must still be determined to localize it within the antigen’s primary structure. In a few unexpected situations, epitopes have been assigned to an enzyme’s active site without sequence information when the antibody was found to block catalytic activity (6). However, results of this sort may not always be reliable, since an antibody can also inhibit enzyme activity by noncompetitive mechanisms.


Protein Antigen Epitope Mapping Digestion Buffer Digestion Mixture Digestion Reaction 
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Copyright information

© Humana Press Inc., Totowa, NJ 1996

Authors and Affiliations

  • Philip S. Low
    • 1
  • Jie Yuan
    • 1
  1. 1.Department of ChemistryPurdue UniversityWest Lafayette

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