Epitope mapping using histidine-tagged protein fragments: application to Escherichia coli RNA polymerase sigma 70

We present a simple, rapid, and inexpensive method for mapping epitopes of monoclonal antibodies using His-tagged protein fragments. In essence, four steps are involved: (i) purify overproduced His-tagged protein from inclusion body; (ii) fragment the protein by partial chemical or enzymatic hydrolysis and isolate the His-tagged peptides with a Ni(2+)-chelate column to generate a "ladder" containing a distribution of peptide fragment sizes; (iii) fractionate the isolated peptides by SDS-polyacrylamide gel electrophoresis and probe the ladder by immunoblot analysis; (iv) determine the size of the fragments that do and do not bind the monoclonal antibody using size markers specific to the His-tagged protein being studied. We have applied this method successfully to map the epitope positions of known and new monoclonal antibodies to Escherichia coli sigma 70. We believe this method will find broad application.