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,
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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,
Contact Us for FREE consultation
on peptide selection.