Severn Biotech has been making bespoke synthetic peptides since 1993.Peptides are synthesised using Fmoc chemistry on a solid phase support matrix. The final quality of the peptide product is assured by Maldi-TOF Mass spectroscopy, *Electro-spray MS and RP-HPLC analysis. Quantities normally supplied are within the range 1mg-30g. There is a wide range of synthetic peptide modifications and conjugations available,which can be incorporated into the product as part of the service. Peptides are supplied lyophilised and can also be aliquoted into vials specific to cilents requirements.
Purity Levels and Peptide Purification
The standard purity is > 85% which is sufficient for antigenic peptides for immunological applications. Ultra-pure peptides of > 95% are obtained by semi-preparative and preparative scale C18 reverse phase HPLC; recommended for bio-active and intracellular studies. All peptides are supplied with HPLC profile and Mass Spectromatogram for verification of purity and molecular weight. Net peptide content by *amino acid analysis can also be supplied. (*Available on request)
|Severn Biotech's bench top MALDI-TOF Mass Spec used in Verification of peptides
Additional Modifications to Peptides
- Acetylation - N-terminal
- Phosphorylation - available at serine, threonine and tyrosine residues
- Biotinylation - N-terminal or C terminal via lysine (both primary amine)
- Cyclisation (disulphide or cyclic formation)
- Fluorescent labelling ( Flourescein, Cy-Dyes, etc )
- Carrier Protein Conjugation ( KLH, BSA etc )
- Farnesylation , Thioester formation etc
- Hexanoic acid,Octanoyl additon etc
- Isotope labelling (Non radioactive)
Many other modifications are available upon request.
Conjugation to Carrier Proteins (Used as Antigen or Hapten in Anti-sera production)
Synthetic Peptides can be conjugated to various carrier proteins for example BSA, KLH, PPD, Thyroglobulin etc using either a hetero- or homobifunctional cross-linking reagent.Peptides are coupled via a carboxy or amino terminal cysteine residue to available amines on the carrier protein. In order to facilitate this reaction a Cysteine residue must be either part of the peptide or added to the N or C terminus. The product is lyophilised in sterile water or PBS. For further details please enquire.
Multiple Antigenic Peptides (MAPs used in Producing Anti-Peptide Antibodies)
Traditionally, small peptide fragments are attached to a large, inert carrier protein to generate an adequate immune response. The multiple antigenic peptide approach eliminates the need for a carrier protein. This is replaced by a branched chain poly-lysine core of low antigenicity. To this core can be added 2,4,8 or 16 antigenic peptides. The result being a stable structure with a high ratio of peptide to core. The poly-lysine core is sufficient for attachment to plastic, thereby performing well in direct immunoassays.
Handling Synthetic Peptides
Supply and Storage of Peptides
Custom-made synthetic peptides are supplied lyophilised to avoid degradation of the product. The majority of peptides, if stored tightly sealed and at -20°C in lyophilised form will remain stable for several years. To maintain stability, it is recommended that vials should be allowed to warm to room temperature in a dessicator with fresh dessicant before opening to prevent condensation forming on the product.
Peptides in solution are much less stable. Storage is recommended at -20°C in a buffered solution at pH 5-7. To avoid bacterial degradation, sterile purified water must be used for dissolution or the solution should be filtered using a 0.45m or 02m filter. The peptide stock solution should be aliquoted out before freezing to prevent damage caused by repeated freezing and thawing. After thawing, any remaining peptide in an aliquot should be discarded.
The secondary structure of peptides is often a hindrance to its dissolution, particularly with hydrophobic peptides. Since the formation of this secondary structure may be promoted by salts, the peptide should first be dissolved in sterile distilled or deionised water. At this stage the concentration of the peptide should be kept higher than the required final concentration to allow for addition of other solubilising agents (if required) and buffer salts which should be added only after the peptide is fully dissolved. Peptides containing cysteine, tryptophan or methionine are particularly susceptible to oxidation and should therefore be dissolved in oxygen-free water (prepared by degassing under reduced pressure or by sparging with an inert gas).
If the peptide does not dissolve in pure water, the following measures may be taken:
- Sonication. This may help to break up any particles and increase the rate of dissolution.
- Dilute aqueous acetic acid or ammonia(10%v/v) added dropwise to basic or acidic peptides respectively.
- If the above methods do not succeed in dissolving the peptide, addition of a chaotropic agent such DMF, Urea or Guanidium hydrochloride may be helpful. Addition should be dropwise until the peptide dissolves. These agents may interfere with biological systems so should only be used if the peptide is being prepared for analytical purposes.Salts may need to be removed by dialysis prior to use in biological systems.
Amino Acid Codes used in Peptide Synthesis
Peptide Ordering / Enquiry Link ]
Enquiries and Ordering
Please use the 3 letter or single letter Amino Acid code when making enquiries or ordering synthetic peptides. Please indicate the quantity and level of purity required. Should you need any help or advice we will be happy to answer any questions you may have.
Order Enquiry Example
A Peptide Sequence is written from the "N"(amino) terminus to the "C"(carboxyl) terminus.
GKYTVALMDQRRSTGC 16 amino acids Required Quantity 10mg >95% Purity.
Price Quotation Estimate
Please supply the peptide sequence required ,should you need a price estimate quotation for the molecule quickly.Please submit this in the notation above and we will contact you immediately to discuss your enquiry..All enquiries are treated with the strictest confidence and no information supplied will be used or forwarded to third parties. Severn Biotech is happy to engage in confidentiality agreements where the client feels the molecular information is their Intellectual Property. For further details please enquire.
Additional Services From Severn Biotech
Anti-Peptide Antibody production including ELISA Testing
Severn Biotech provides a fully flexible service to provide polyclonal and mono-clonal antibodies. Typical immunisation protocols extend over 77 days and include pre and post immunisation ELISA testing of sera throughout the period. The service is UK home office approved and US NIH compliant. For further details please enquire
Affinity Purification Service to include ELISA Testing
Affinity columns are made by binding a specific peptide or protein to Thio-linkTM Gel. This is then used to remove or capture the specific antibody to that peptide or protein from the anti-sera. This is then eleuted from the column as the purified sera effectively removing the non specific antibody molecules.Purified sera is then verified by ELISA testing the eluted purified material against the antiigen
Custom made Affinity Purification Columns
Affinity columns are made by binding the protein or peptide of interest to Thio-linkTM Gel which is housed within a poly-propylene tube.The bound material is specific to the anti-sera to be purified and used to capture then elute the molecules of interest. Colulmns can be washed and re-used to batch the sera being purified.The bound hapten is irreversibly attached but can be re-used several times.For long term storage use a PBS buffer solution containing sodium azide to preserve the column integrity.End caps are supplied to keep the column from drying out. For further details please enquire
Peptide Design and Antigenicity Prediction.
Severn Biotech has many years experience in designing peptides particularly from BLAST search data. Not every sequence is suitable for use in anti-sera production and specific epitopes are often non viable. We can aid your decision making in determining the best peptide sequence to synthesise which will help predict a specific anti-body to the protein of interest. For further details please enquire.
Protein Sequencing using Edman degradation
N terminal sequencing of proteins and pepides is attained using edman degradation.This enables sequence data to be used in protein determniation. Sequence data is very useful in designing DNA probes and primers. Protein material can be supplied from PVDF mambranes,stained Gels and blots, liquid fractions from HPLC and freeze dried material. For further details please enquire.
Amino Acid Analysis
Amino Acid analysis is achieved by ion exchange methods and HPLC, it is ideal for determining net peptide content for GMP requirements, QC data with peptide synthesis and use in food and drink analysis.
For further details please enquire. / Enquiry Link ]