Peptide Synthesis for Antibody Production
Peptides are synthesized using Fmoc solid phase technology. The most common length of peptide utilized for antibody production is between 10 and 20 residues, but peptides as small as 4 amino acids and as large as 75 amino acids can be made. All peptides used for antibody production come with complete MALDI mass spec and/or HPLC analytical data.
When examining peptides for synthesis and immunization, it is recommended that certain residues and sequences be avoided due to potential synthesis problems. This list includes some of the more common problems:
- Extremely long repeats of the same amino acid (e.g. RRRR)
- Serine (S), Threonine (T), Alanine (A), and Valine (V) doublets
- Ending or starting a sequence with a proline (P)
- Glutamine (Q) or Asparagine (N) at the n-terminus
- Peptides over weighted with hydrophobic residues (e.g. V,A,L,I, etc.)
For additional information regarding the solubility of a peptide sequence, please visit Peptide Solubility
Antigen Design
Open Biosystems has designed a set of powerful algorithms that enables researchers to analyze protein sequences to select the best peptides for antibody production.
- Examine comparative antigenicity based upon actual in vivo titer data from over 13,000 peptide antibodies
- Incorporate important structural motifs that are proven sites for producing high binding antibodies
- Built-in peptide tutorial for synthesis and solubility
- Specificity determination through curated BLAST analysis
For more information on Open Biosystems' antigen design, please visit Antigen ProfilerTM. Below is a list of general rules to use when designing a peptide for antibody production. General Rules for Peptide Design |
Hydropathy | Make sure you have more hydrophilic than hydrophobic residues, and avoid large the presence of repeated, bulky hydrophobics (e.g. W and Y) |
Balanced Charge | The peptide should have an overall charge close to neutral |
Hydrophilic Terminal Residues | End sequences with hydrophilic residues if possible as favorable side groups will promote increased solubility along with free alpha reactive groups. Terminal ends are highly exposed to their environment |
Size of Peptide | Try to limit the size of the peptide to 25 residues or less. This will limit the amount of salvation shell formation |
Contiguous Residues | Limit the number of contiguous charged or hydrophobic residues as they can “isolate” a portion of the peptide leading to increased solubility problems |
Blocking | Consider amidation and acetylation effect on solubility. If a peptide has more hydrophobic residues than hydrophilic residues, then do not cap the alpha residues as free alpha amines and carboxylic acids are attracted ionically to water |
Capping | Do not cap hydrophobic terminal residues if possible. If you have to, then try to leave at least one uncapped to promote increased solubility |
Multiple Residues | Avoid the presence of multiple cysteines and methionines as they can oxidize or form dimers with other peptides (cysteine) |
Terminal Residues to Avoid | Avoid ending sequences in proline, valine, isoleucine, tryptophan, tyrosine, and phenylalanine |
Doublets | Avoid proline, alanine, valine, threonine, or serine doublets for synthetic purposes |
Hydrophobic Sequences | If a sequence is highly hydrophobic with no alternatives, then consider artificially adding a terminal cysteine and conjugating through it |
Glycines | Due to the nature of glycine and its lack of side group, do not count it as a hydrophobic residue unless contiguous stretches exist |
