Epitope Mapping Protocols pp 77-86

Part of the Methods in Molecular Biology™ book series (MIMB, volume 524)

Proteolytic Fragmentation for Epitope Mapping

  • Maria R. Mazzoni
  • Francesca Porchia
  • Heidi E. Hamm
Protocol

Summary

The use of antigen fragments generated by specific proteolytic cleavage is a relatively simple “library” approach for epitope mapping in which possible overlapping fragments are screened with the antibody on Western blots. Proteolytic fragmentation with numerous proteases having different cleavage specificites can be carrried out on native and denaturated proteins, generating a small and large number of fragments, respectively. To determine the antigenic site of a monoclonal antibody, we have examined the limited proteolytic digestion of the transducin α -subunit with four different proteases and detected antibody binding to fragments by Western blot. Using this approach, the epitope for this antibody was localized within the amino-terminal 17 residues of transducin α -subunit.

Key words:

Epitope Monoclonal antibody Protease Proteolytic fragmentation Western blot 

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Maria R. Mazzoni
    • 1
  • Francesca Porchia
    • 1
  • Heidi E. Hamm
    • 1
  1. 1.Department of Psychiatry, Neurobiology, Pharmacology and BiotechnologyUniversity of PisaPisaItaly

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