Double-check that the correct species and amino acid sequence have been identified.

Domains will be present in other proteins and should therefore be avoided in the sequence of the immunogen as they will increase the likelihood of cross reactivity with other proteins.

Not all areas of the protein, such as the center of a protein or transmembrane regions, are accessible to antibodies.

Accessible regions of the native protein are regions exposed on the surface of the protein and in contact with the aqueous environment. Computer programs are available that assign a “hydrophilic index” to each amino acid in a protein and then plot a profile. A hydrophilicity program is contained within most protein database systems, such as ProtScale.

Avoid long chains of hydrophobic residues.

Avoid complex regions such as alpha helices and beta sheets. Aim instead for flexible regions.

Avoid Glutamine and Aspartamine. These can change to Glutamic and Aspartic acid, causing hydrogen bonding between peptides.

Try to incorporate proline and tyrosine residues. These confer some structural motif to the immunogen, which is likely to be found in the native protein.

Avoid regions known to be post-translationally modified as they may mask the site of antibody recognition.

Avoid Cysteine residues, as they can form disulfide bonds between other Cysteines to form intra- and inter-chain Cysteine.