- How should I store and handle my peptides?
Because peptides are hygroscopic, warm up the peptide to room temperature in a desiccator prior to opening and weighing out the portion of the peptide to be used. Failure to do so can cause condensation to form on the product when the bottle or vial is opened and greatly reduce the stability of the material. Weigh out quickly in a clean environment only the needed quantity of the peptide to be used to minimize contamination and reseal container tightly. Store all unused peptide at -20° C or less in a sealed container with desiccant. Storage under these conditions will reduce and prevent bacterial degradation, secondary structure formation, and oxidation for up to several years. Sequences containing cysteine, methionine, tryptophan, asparagine, glutamine, and N-terminal glutamic acid will have a shorter shelf life than other peptides.
Sonication is often helpful to dissolve the peptide. In general, sterilized water is the preferred solvent, but acidic peptides will dissolve in a basic buffer and basic peptides in an acidic buffer as determined by the amino acid sequence of the peptide. Peptide sequences containing Cys, Met, and Trp require oxygen free solvents as they are especially prone to oxidation. Peptides in solution are much less stable than lyophilized form and will remain viable for a much shorter period of time. As such, it is not recommended to store peptides under these conditions, but if absolutely unavoidable, the peptides in solution should be kept at -20° C using sterile buffers at pH 5-6 to prolong shelf life. The best way to avoid running into this issue is to ask us to aliquot the peptide.
- What peptide purity is recommend?
Immunological grade and
Antigen for antibody production.
ELISA standard for measuring titers of antibodies in antisera.
Competitive elution chromatography.
Peptide array production.
> 80% purity
Non-quantitative enzyme-substrate studies.
Non-quantitative peptide blocking studies.
Coupling to resins for affinity purification.
Coating of tissue culture plates for cell attachment.
Protein electrophoresis applications.
Standards for quantitative ELISA and RIA protocols.
Quantitative receptor-ligand interaction studies.
In vitro bioassays.
In vivo studies.
Quantitative enzyme studies.
API (Active Pharmaceutical Ingredients)
X-Ray crystallography studies
- What is peptide purity and how does it differ from peptide content?
The reported purity of a peptide is a percentage of the target sequence as determined by HPLC. The impurities found in a peptide will include deletion sequences, truncated sequences, and incompletely deprotected sequences. Peptide content can be determined in a few different ways depending on the sequence, and is a measurement of the native peptide present versus the non-peptide components.
- How do you conjugate my peptide to a carrier protein for antibody production?
When producing peptide-antibodies, it is important to use a carrier protein to elicit a better response from the animal. Commonly used carrier proteins are BSA and KLH, with KLH preferred for its higher immunogenicity. Peptides coupled with BSA will generate antibodies against both the peptide and the BSA, which could result in incorrect ELISA readings.
For coupling carrier proteins to peptides, we recommend incorporating an N terminus cysteine for this reaction due to the high efficiency and proven effectiveness. For coupling through cysteine, we use MBS (maleimidobenzoic acid-N-hydroxysuccinimide ester) as a crosslinker. The initial step is to activate the protein with MBS, and then remove the excess crosslinker by SEC. This activated carrier protein is then coupled to the cysteine in the peptide through a disulfide bond with an efficiency of >95%. The only impurity in this reaction is salt formation, which we remove through gel filtration.
As an alternative to coupling through cysteine, we can additionally couple the carrier protein of choice through an amino link. The coupling efficiency of this reaction is generally around 80%. For carrying out this reaction, glutaraldehyde is used to crosslink the amino groups in both peptide and carrier protein. The most undesirable part of this reaction is the fact that protein-protein and peptide-peptide crosslinking can occur, and for this reason, the most important step is to insure that the proper concentration of crosslinker.
- How are peptides synthesized?
The crucial link in peptide synthesis is the peptides bond formed by condensation of the alpha-amino group of one amino acid and carboxyl group of another amino acid. Amino acids contain a central carbon atom surrounded by hydrogen, an amino group, a carboxyl group, and a side chain group, designated as R. This R group is what defines each individual amino acid and determines the physical and chemical properties of the amino acid. Certain R groups can interfere with the formation of the peptide bond, and, therefore, it is necessary to orthogonally protect the functional side chains of certain amino acids. Different protecting groups are available depending on the type of chemistry used and the application.Synthetic peptides differ from natural protein synthesis on ribosomes in that the synthesis is conducted from the C to N terminuses. In solid phase peptide synthesis, the first amino acid is covalently linked to a solid support with the alpha amino group protected by either an Fmoc or t-Boc moiety. Treatment with a deprotection agent (piperidine for Fmoc, TFA for Boc) frees the alpha amino group in preparation for coupling the next amino acid in the sequence. The next amino acid is activated by one of several reagents and a peptide bond is formed.
This freshly coupled amino acid is protected on its alpha amino group as well, and in this way prevents double addition of amino acids into the sequence. This stepwise addition continues until the desired peptide length is obtained. After the last amino acid has been added, it is necessary to perform one additional deprotection step to remove the last moiety on the N terminal amino acid. Once the synthesis has been completed, the peptide must be removed from the solid support. This is accomplished by adding an appropriate acid (TFA for Fmoc, HF for Boc) as well as scavengers to collect protected functional groups.
- Are there any restrictions on what can be produced?
In general, peptides of more than 35 amino acids can be problematic. Solubility is one of the most important factors to consider when designing a peptide as it will affect the purity attained since HPLC is performed in solution. The more hydrophobic residues in the sequence, the less soluble the peptide is. Certain sequences can also aggregate which will lead to difficulties in production.
- How are my peptides shipped?
All peptides are shipped directly to the end-user by FedEx overnight service. On the day of the shipment, an e-mail is sent to the end-user confirming the shipment and providing a FedEx number in order to track the package if needed.
Each peptide is shipped at ambient temperature and accompanied with desiccant to absorb excess moisture. Various tests have revealed shipping under these conditions has no impact on the viability and shelf-life of the peptides. However, upon receipt of the product by the end user, it should be stored immediately at -20° C or lower.
- What is the difference between catalog and custom peptides?
Catalog peptides are commercially available, published sequences. Commonly used catalog peptides are frequently produced in bulk at purities exceeding 95%, and they are generally kept in-stock. Producing these peptides in larger quantities allows companies to sell them at lower prices due to the bulk process involved. Custom peptides, on the other hand, are not public domain. Certain changes or modifications to catalog peptides must be ordered as a custom order for this reason. When ordering a custom peptide, the two most determinate factors are purity needed and sequence length. The time length involved in producing a custom peptide at CPC is generally 2-3 weeks.
- What type of QC will be done on my peptides?
Every peptide (including crude) is shipped with HPLC and MS. A bonus that Peptides America provides with its QC package is a recommended solubility test on each peptide. This solubility test utilizes solvents that are applicable for your experiment (water is the first choice), and can be customized according to the customer’s specifications. The test will save you time and effort in trying to determine the solubility and will save peptide because you will not need to use a small sample for testing if you do not have the ability to lyophilize the peptide
- What kind of peptide modifications can Peptides America produce?
Peptides America can produce all commercially available modifications with the exception of radiolabeling. Peptides America is happy to provide you with answers to any of your technical questions concerning modification. For a list of peptide modifications and some intended uses, please visit the peptide modification page.
- Can Peptides America aliquot my peptide?
Peptides America can indeed aliquot any order to your specifications. Through aliquotting your order into smaller quantities, this service will allow you to store and access your peptide with ease and gives you the ability to standardize your protocols. Perhaps most important, the peptide will enjoy greater stability in the long run by preventing contamination from frequent opening of the container. Repeated thawing/freezing and opening/closing of the container will invariably introduce moisture into the system. This could lead to bacterial degradation, peptide oxidation, and aggregation, lowering the purity of your peptide and causing loss of continuity in future experiments. Weights for aliquots 1 mg and over will be determined by our balance scale. All aliquots less than 1 mg will be aliquoted volumetrically for more precision than our balance offers.