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1. Ensure that the correct species and protein sequence have been identified
2. Use OMIM and LocusLink to establish domain regions. 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. Also certain areas of the protein may be inaccessible to antibodies i.e. centre of globular protein or transmembrane regions.
3. Select the peptide immunogen from an accessible region of the native protein. Often these are regions exposed on the surface of the protein in contact with the aqueous environment (hydrophilic). Computer programs are available that assign a "hydrophilic index" to each amino acid in a protein and then plot a profile. The regions of hydrophilicity can then be seen.
4. A hydrophilicity program is contained within most protein database systems, some of these are:
- ProtScale
- PlotStructure
- PepPlot (good overview of what all the different plots mean)
5. Avoid long chains of hydrophobic residues.
6. Avoid complex regions such as alpha helices and beta sheets, instead aim for flexible regions.
7. Avoid multiple Glutamine residues as this can result in hydrogen bonding between peptides
8. 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.
9. Avoid regions known to be post-translationally modified as they may mask the site of antibody recognition.
10. The addition of a terminal cysteine residue to the peptide sequence allows conjugation of the peptide to carrier proteins, such as KLH or BSA using the Sulfo-SMCC (Sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate) linker (PIERCE).
11. A terminal Cysteine residue also allows straight-forward immobilisation of the peptide on the SulfoLink affinity matrix (PIERCE), if the resultant antibody requires affinity purification.
N.B. If the peptide is derived from the N terminal of the protein the cysteine should be added to the C terminal end of the peptide and vice-versa. If the peptide is derived from an internal protein sequence then the cysteine can be assigned arbitrarily.
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