by
experience
Peptide synthesis and coupling to the carrier, including our assistance in the selection of suitable sequences
Peptide design - there are many important factors to consider when choosing a suitable part of a protein sequence for successful antibody production. AgriSera will assist and offer help by experienced peptide chemist.
Development of a peptide target conserved in all proteins from protein family under investigation as well as peptides for antibodies specific for chosen proteins from protein family. Produced antibody will recognize all proteins within the family. Please, inquire.
More information about the approach to develop global target peptide, can be found in the following
article: "Analyzing photosynthetic complexes in uncharacterized species or mixed microalgal
communities using global antibodies" Campbell D., Cockshutt A., Porankiewicz-Asplund J. (2003)
Physiologia Plantarum 119:322-237.
This article can be downloaded here. global_antibodies.pdf
General information:
- Peptide length - standard 4-14 amino acids. Synthesis of longer
peptides is also possible.
Terminal cysteine will be added to make peptide orientation on the carrier possible.
The peptides are synthesised by Solid Phase Peptide Synthesis, using Fmoc-Chemistry. - Peptide modification - acetylation, biotinylation and fluorophore
coupling. For other modifications, please inquire.
- Coupling to the carrier, usually done through terminal cysteine to allow peptide orientation
on a carrier: KLH, BSA or other.
- Delivery time - 3 weeks.
- Quantity - up to 25 mg within the same price or 100 mg
- Purity levels - from immunograde purity (> 70 %) which is sufficient
for generating or testing antibodies, to higher purity (+ 95 %) required in enzymological and
biological activity studies and other applications.
- Quality control - includes HPLC and mass spectrometric analysis. On special request we can offer amino acid analysis and sequencing.
Most common carrier proteins:
KLH- keyhole limpet hemocyanin is a copper-containing protein, isolated from the hemolymphs of the mollusk. It exists in five different aggregated states (in Tris buffer pH 7.4), which readily dissociate with moderated pH change. Subunit molecular mass ca. 450 kDa.BSA- bovine serum albumin, subunits of 67 kDa. Popular protein used in immunoassays to block non-specific binding sites. It should not be used as carrier if future assays involve BSA (for instance as blocking agent).
Question: I would like to do the conjugation myself using glutaraldehyde,
what do AgriSera recommend?
Glutaraldehyde coupling will result in on of most stables linkages, however,
glutaraldehyde bridge can often form a portion of an epitope recognized by the
immunized animal. Therefore immune serum should always be screened in the
presence of control- other glutaraldehyde-coupled peptide, since in some cases,
positive serum can be mistaken from the reaction of antibodies against
glutaraldehyde.
Glutaraldehyde cross-linking is useful when peptide contains only one free
amino group. In case of more free amino groups, large, multimeric complexes
will be formed. In such cases other coupling chemistries should be applied.
This will not allow peptide exposure on the carrier protein.
Disadvantages of using anti-peptide antibodies:
- Not all anti-peptide antibodies will recognise the epitopes in the native protein.
- High concentration of anti-carrier antibodies present in the antibody pool may lead to increased background levels.
AgriSera is offering polyclonal, anti-carrier antibodies (anti-KLH and anti-BSA).
Alternative MAP system, no carrier protein involved:
MAP- multiple antigenic peptide. This system uses peptides attached at many positions to a polylysine core. MAPs containing 2 to 16 copies of synthetic peptide molecule can be produced.
Advantages of MAP system:
- Maximisation of antigen concentration (95 % of MAP is the antigenic peptide)
- Suitable for epitope mapping
- Alternative if KLH or BSA conjugates fail.
- Possible lower probability of obtaining antibodies in some cases, compared to the system which utilize carrier proteins.
Last changed: 2009-04-02 by WebMaster