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• Peptide Synthesis

Sample protocols

Peptide Design Considerations

Recent advances in solid phase peptide synthesis have greatly improved the throughput of peptide synthesis but success depends heavily on the primary sequence of the peptide to be synthesized. The following points should be considered in choosing a peptide of synthesis:

1. Peptides with multiple amino acid repeats couple poorly in solid phase synthesis strategies and result in poor yields.
2. Peptides with C-terminal Ala, Leu, Ile, Val, Glu, and Ser often aggregate.
3. Avoid Gln or Pro as N-terminal residues.
4. Side chain reactions can occure with the following amino acid pairs: Asp-Asn, Asp-Gln, Asp-Gly, Asp-Ala.
5. Multiple arginines can cause adduct formation in crude peptides.
6. Peptide yields are inversely proportional to the number of hydrophobic residues

There are a number of methods to maximize the chances of success in peptide synthesis and we encourage investigators to discuss planned peptides with our facility scientist.

Tips For Designing an Antigenic Peptide

Most naturally offering proteins have hydrophilic surface residues and buried hydrophobic residues. The best epitopes for a protein in its physiological state are found on the surface of the protein and often have a high degree of mobility. The N- and C-termini of proteins are generally surface-oritiented and make good epitope candidates. There are a number of programs that use algorithms for assigning values of hydrophilicity, surface probability, flexibility, antigenic index and amphiphilicity of a protein sequence. These programs are very helpful in choosing candidate epitopes. In addition, the following points should be considered

1. Exposure on the Surface of the Protein: A peptide composed mainly of hydrophilic amino acids is, most likely, to be located on the surface of the intact protein molecule. This is deduced by plotting the hydrophilicity plot for the protein. Such a peptide would be a good candidate for producing antibody.

2. Immunogenicity: Some amino acid residues seem to be more immunogenic than others, since they are found in relatively high frequency in antigenic epitopes. These are, in decreasing order: His > Lys > Ala > Leu > Asp > Arg

3. Length of the Peptide: The length of the antigenic determinant is about six amino acids. So the minimum size for a peptide is 6 amino acid residues. Generally, peptides containing 8 -15 residues lead to greater success.

4. Lack of Modification(s): Peptides which show phosphorylation or glycosylation sites should not be chosen for immunization.

5. Crosslinking (Conjugation) of the Synthetic Peptide to Macromolecules: Peptides with M.W. of 700-1,500 give better response when attached covalently to large carrier proteins. Most commonly used carrier proteins are: keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), tetanus toxoid (TT), ovalbumin, myoglobin, etc. Several chemical methods of conjugation are available. Conjugation from either N- or C- terminal would presumably be preferable as this will not alter the side-chains of the AA residues. Multiple antigen peptide (MAP), developed by Tam et al., does not require conjugation. These MAPs have become very popular for immunization over the past few years. Peptides linked covalently from their C-terminals to polyamide resin supports also do not require conjugation.

Handling of Peptides

Peptides are usually supplied as a fluffy, freeze-dried material in plastic serum vials. They should be stored at -20 degrees C as dry powders. After reconstitution, they should be used as soon as possible to avoid degradation in solution. Unused peptide should be aliquoted into single-use portions, relyophilized if possible, and stored at -20°C. Repeated thawing and refreezing should be avoided.

Peptides should be reconstituted in distilled water to generate stock solutions that can be diluted into a buffer solution of choice. Highly charged peptides and hydrophobic peptides are often not soluble in water. Positively charged peptides must be dissolved in acidic solutions (i.e. 10% acetic acid) and negatively charged peptides must be dissolved in basic solutions (i.e. 10% ammonium bicarbonate). Note: do not use basic solutions for cysteine containing peptides. Other solvents such as acetonitrile, DMSO, DMF, or isopropanol may be used to dissolve peptides for stock solutions that can be further diluted in aqueous buffers for use. For peptides that tend to aggregate chaotropic agents such as urea or Guanidine hydrochloride may be used.