Proteolytic Fragmentation for Epitope Mapping

  • Maria R. Mazzoni
  • Nikolai O. Artemyev
  • Heidi E. Hamm
Part of the Methods in Molecular Biology™ book series (MIMB, volume 66)

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.

Keywords

HPLC Glycine Electrophoresis Ketone Trypsin 

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Copyright information

© Humana Press Inc. 1996

Authors and Affiliations

  • Maria R. Mazzoni
    • 1
  • Nikolai O. Artemyev
    • 2
  • Heidi E. Hamm
    • 3
  1. 1.Institute Policattedra di Discipline BiologicheUniversity of Pisa College of PharmacyPisaItaly
  2. 2.Department of Physiology and BiophysicsCollege of Medicine, University of IowaIowa City
  3. 3.Department of Physiology and BiophysicsCollege of Medicine, University of Illinois at ChicagoChicago

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