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Custom Peptides and Antibodies

  • How do I choose peptide endings appropriate to my studies?

    The choice of peptide endings often falls simply to the question: am I happy with the default of a free amine group at the N-terminus of the peptide, and a free acid group at the C-terminus of the peptide, or do I need something different? A good example of where something different is needed, is where the peptide is a natural hormone or an analog of a natural hormone with an amide group at its C-terminus, such as Substance P. In a case like that, the only reason to vary from having the peptide made with a "natural" amide C-terminus would be to examine the effect of placing an unnatural free acid group on the C-terminus.
    Another common situation is where the peptide represents an internal portion of a protein sequence, i.e. does not arise from the natural N-terminus or C-terminus of a protein. It may be best in such cases to "block" the ends of the peptide, such as by having the C-terminus made as an amide, and having the N-terminus blocked by acetylation. The peptide would then be indicated as having the structure:

    Acetyl-(Peptide Sequence)-amide.
    By using this design, the following is achieved:

    1. It avoids the unnatural introduction of a charged group (amine or acid) at a site in the peptide, where the same site in the parent protein has no such charged group. Blocking the ends like this can be beneficial because the peptide is then more likely to behave like, or be recognized, as if it were a part of the whole protein from which the sequence was chosen.
    2. The peptide will be more resistant to breakdown resulting from the actions of exopeptidases, which attack the peptide from the ends. It would then persist longer in the bioassay system, such as an assay involving living cells or biological extracts which contain peptidases.

    Not all effects of blocked ends are helpful, however. Blocking charged groups on the ends of a peptide decreases the solubility of the peptide, maybe to the extent where solubility becomes a limitation on the effective concentration of that peptide which can be obtained in solution. It may even make the peptide so hard to dissolve that its usefulness is compromised. Thus, a decision may need to be made about what is more important: solubility, or the closeness of a peptide's structure to resembling the protein from which its sequence was derived?
    Similar issues arise each time a new peptide study is devised. For example, in the study of cytotoxic T cell epitopes, it is appropriate to have short peptides made with free amine and free acid endings, because they are the natural endings of peptides which have been processed intracellularly from whole proteins. Natural helper T cell epitopes are longer than cytotoxic T cell epitopes, and even though in nature helper T cell epitopes have free ends, relatively short end-blocked synthetic peptides may function better in helper T cell assays than peptides with free ends. The reason may be related either to the synthetic peptide not being made with an ideal length, or it may be related to the amount of time the peptides persist in cultures before being broken down. The lengthened amount of time that end-blocked peptides have, to begin exerting a biological effect in a culture, may be more important than their ability to exactly mimic a natural epitope.
    Other endings which may be chosen for specific purposes include:

    1. Adding a biotin group at one end of a peptide (usually the N-terminus) as a convenient way to capture the peptide via a high affinity noncovalent interaction;
    2. Adding a Cysteine residue at one end of a peptide to allow covalent capture under mild conditions; or
    3. Adding a fluorescent group, to allow tracing of the peptide.

    Please contact Mimotopes if you need help in designing a set of peptide endings appropriate to your project. Our Peptide Support Group would be only too pleased to assist you with advice, based on our combined experience.

  • How long would it take to raise antipeptide antibodies?
  • What purity is offered for custom synthesized peptides and how is this purity characterised?
  • What are MAPs and how can they be useful?
  • Does Mimotopes supply GMP grade peptides?
  • Is there any way of determining which region of a protein will be favourable for raising antibodies?
  • I require a fluorescent label peptide, which fluorescent labels do you offer?
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