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FAQ PEPTIDE SYNTHESIS
Some frequently asked questions about polyclonal antibodies (click on the question to see the answer):
The purity required depends on the application. Peptides with more than 70% purity are recommended for screening purposes and polyclonal antibody production. Peptides with more than 80% purity can be used for qualitative biochemical applications, e.g. enzyme-substrate studies or production of affinity resins. For all quantitative enzymology and biological assays we recommend peptides with more than 95% purity.
We routinely synthesise peptides between 3 and 90 amino acids long. Sequences with less than 3 residues may be difficult to cleave from the resin and therefore to purify. Peptides longer than 90 amino acids are often more difficult to synthesise with high purity.
- Several factors can affect the synthesis and purification of a peptide including amino acid succession and overall composition, peptide length and hydrophobic stretches.
- Highly hydrophobic peptides including residues such as Leu, Val, Ile, Try, Phe and Met may be difficult to purify and to solubilise in aqueous solutions. As a rule of a thumb, we recommend one charged residue for every five amino acids.
- Highly pure peptides with multiple Cys, Met or Try residues can also be difficult to obtain as these residues are prone to oxidation/hydrogen bonding. Producing these peptides may cause a slight delay due to repeated synthesis attempts and/or purifications.
- Several Ser and/or Asp residues in a row may result in a product of lower purity. Multiple Pro in the sequence may undergo cis-trans isomerisation which may result in lower apparent purity. For peptides with a N-terminal Asn or Gln, we recommend to remove, add or substitute with another amino acid in order to obtain a higher quality product.
We are experts in synthesising phosphorylated sequences including phospho-serine, phospho-threonine and phospho-tyrosine residues. To increase the coupling efficiency, the phosphorylated residue should be located no more than 10 amino acids away from the N-terminus. However, we are not able to synthesise peptides with more than one phospho-residue.
The turn-around time depends on the peptide quantity and purity required. Most peptides (up to 80% purity, 5-25mg) are synthesised within 2-3 weeks. Delays can occur for longer peptides (25+ residues) and difficult sequences, for example very hydrophobic sequences at high purity (>95%).
Every endeavour is made to ensure the quality of the synthesised peptides. If a lower purity or quantity is obtained, you will be charged accordingly.
- Peptides used to immunise an animal (usually 12-18 amino acids) require fusion to a carrier protein as the molecular weight of the peptide is generally not sufficient to elicit an immune response. When raising anti-peptide antibodies which should recognise the native protein, coupling of the peptide to the carrier protein is very important and should correspond to the natural representation of the peptide in the protein.
- If the immunogenic region of the peptide is N-terminal, it should be coupled through its C-terminal amino acid and vice-versa. If the immunogenic region is internat, either end can be targeted (and preferably the less immunogenic).
- Amidation of the C-terminus and acetylation of the N-terminus is recommended for internal peptides to avoid the introduction of non-natural charges (positive charge at the N-terminus or negative charge at the C-terminus). For example, the C-terminus of an internal peptide (fused at its N-terminus) should be amidated as this functional group (CONH2) mimics best the peptide bond between two amino acids.
The best known carrier proteins are keyhole limpet hemacyanin (KLH), bovine serum albumin (BSA), ovalbumin (OVA) and bovine thyroglobulin (THY). KLH is the most commonly selected carrier due to its high immunogenicity and will generally not be used as a blocking reagent in experimental assays.
The solubility of a peptide depends on its composition.
The peptides are supplied lyophilised and should be stored at –20°C.
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