Epitope Mapping by Chemical Fragmentation

The use of antigen fragments generated by specific chemical cleavage is a relatively simple “library” approach for epitope mapping in which overlapping fragments are screened with antibody on Western blots. It is widely applicable insofar as it is not restricted to recombinant antigens only, but the amino-acid sequence of the antigen must be known. It cannot be used for highly assembled epitopes, which will be affected by the denaturing conditions of cleavage and SDS-PAGE, as well as by fragmentation itself. Chemicals that cleave proteins at uncommon amino acids are used to produce fragments; cyanogen bromide (CNBr) cleaves C-terminal to methionine residues (1 ), nitrothiocyanobenzoic acid (NTCB) at Cys (2 ), iodosobenzoic acid (IBA) at Trp (3 ), and formic acid between Asp-Pro bonds (4 ). The fragments on Western blots can often be recognized unequivocally using M _r s predicted from the sequence.