What are my options for generating antigens? What are the tradeoffs?
Many believe that the best antigens are native proteins because they are correctly folded and provide optimal templates for antibodies that might recognize different kinds of determinants such as conformational (good for immunoprecipitation) and linear (good for Western blotting) epitopes. In practice, it is difficult or impossible for most labs to purify adequate amounts of native protein from biological mixtures. Therefore, antigens are generally prepared by recombinant technologies (ie, expression of 6XHIS-tagged proteins in bacteria) or by chemically synthesizing peptide antigens. Peptide antigens have a couple of obvious advantages. They are easy to generate and by definition define the antibody binding site on the target protein. If you want to target a particular defined epitope within a protein, a peptide antigen is the obvious way to go. Peptides work best if they are greater than 12 amino acids in length, and must be conjugated to an appropriate carrier protein (eg, BSA or KL
