Peptide sequence selection is likely the most difficult step in the development of anti-peptide antibodies. Although the design of peptides for use as antigens is not exact, The following summary highlights some important points that should be considered in the design of a peptide. If you would like BSI to help you in designing your peptide, click here to request for sequence analysis or visit our Gizmo for online interative analysis. Remember! You design. We can Help!
1. Examine N– and C-terminus of the protein. If they are hydrophilic, they often constitute an excellent choice.
2. If both N– and C-terminus are suitable, choose internal hydrophilic region of the protein. Hydrophilic sequences have the best chances to reside at the surface of the protein and therefore to be accessible to the antibodies.
3. Avoid possible glycosylation or phosphorylation sites
4. Avoid a series of hydrophobic amino acids such as alanine, tryptophan etc.
5. Short synthetic peptides are quite flexible and are unable to mimic alpha helical sequences. However, run algorithms for predicting secondary structures and avoid regions which are strongly predicted alpha helical prior to peptide selection.
6. A peptide range between 10-20 amino acids is recommended. Shorter sequences have fewer chances to include good peptides, longer peptides tend to make stable secondary structures which are not necessarily the same in the protein of interest. Longer peptides also increase the chances of cross-reactions with other proteins.
7. A BLAST homology searches of the selected peptides against proteins of the organism obtained from in order to exclude unwanted cross-reactions to other proteins as much as possible. Less than 4-5 identical amino acids in a continuous stretch are required to exclude a cross-reaction.
8. Carrier protein coupling strategy needs to be taken into account. Alternatively, use the MAP strategy for synthesis of the peptide (not suitable for peptides from the C-terminus).
-Peptides from the C-terminus of the protein should be linked to the carrier through the N-terminus.
-Peptides from the N-terminus of the protein should be linked to the carrier by their C-terminus.
-Internal peptides should be coupled at the less hydrophilic end.
-N-terminal coupling is specific only if the peptide does not contain lysine residue.
-C-terminal coupling is specific only if the peptide does not contain aspartyl or glutamyl residues.
-If no specific coupling can be done, add cysteine at one end of the peptide. The only restriction is there should not be any other cysteines in the sequence.
9. Conjugation of a peptide to a carrier protein containing multiple epitopes, such as Keyhole-Limpet Hemocyanin (KLH) or Bovine Serum Albumin (BSA), are most common. KLH is preferred since it is more antigenic in the majority of animals. BSA is often used as a blocking reagent in assays, thus an anti-peptide antibody raised against a BSA conjugated peptide will show some specificity towards the buffer reagents. This can result in a false positive signal.
10. MAP is simply an alternative to KLH conjugation. It is recommended only for shorter peptides located either internally within a protein or at the N-terminus. KLH conjugation is recommended for a peptide at any location in a protein and when there are a cysteine residues within the peptide sequence.
11. Use at least 2 animals for each peptide since there is some genetic variability between animals.
12. Use more than 1 peptide for each protein in order to increase the chances for antibodies to recognize the protein.
The services for the separation of the antibodies produced against the individual peptides are available at Bio-Synthesis, call 800-227-0627 or write to us at support@biosyn.com for FREE consultation on peptide selection.
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