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Custom Peptide Synthesis Frequently Asked Questions
(I) Before Placing Your Order.
- What purity of peptide do I need for my peptide order?
The minimal requirement of peptide purity is dependent on your application or assay. Please refer to our summary Table for recommended purity. In general, we recommend order peptides with > 90% purity.
- At what scale you should have your peptides synthesized?
At Peptide 2.0, we recommend all custom peptides be synthesized at 5 mg scale. There are no major difference in cost between 5 mg and 1 mg scale. Large lots can be fulfilled upon request.
- Is there any limitation on the length of peptides that can be achieved?
We can offer custom service for peptides ranging from 2 to up to 100 amino acids. Standard solid phase procedures can produce peptides in range from 5 to 50 amino acids in length. For long peptides over 50 amino acids, we use convergent peptide synthesis approach to link two peptides in solution. Please note, the efficiency for synthesis and purification for longer peptides will be much lower than usual, and the production time may be substantially longer.
- Can custom peptides be modified based on customers. request?
Peptide 2.0 provides peptide modifications such as acetylation, biotinylation, phosphorylation and attachment of fluorescent dyes or quencher pairs. Please contact our technical support to discuss your specific needs.
What are most common terminal modifications for synthetic peptides?
The default ends of a peptide are a free amine group at the N-terminus and an acid group at the C-terminus. However, we recommend more .native. modifications for synthetic peptides, namely, blocked N-terminus (i.e. acetyl) and C-terminus (i.e. amide). These modifications eliminate risk of introducing additional charges and make the resulting peptides more resistant to enzymatic degradation of exopeptidases.
- What types of synthesis methods are available at Peptide 2.0?
Our peptide chemistry team is headed by a Ph.D scientist with over 20 year experience in peptide synthesis. The team possesses a complete range of peptide synthesis techniques including solid phase and solution phase synthesis with BOC chemistry and Fmoc chemistry. More importantly, our chemists are skillful in stepwise synthesis, convergent synthesis approaches, and ligation techniques.
- How do Peptide 2.0 quality control synthetic peptides?
With exception of crude peptides requested by customers, which will only be confirmed by MS, all synthetic peptides from Peptide 2.0 will be analyzed by HPLC and MS. HPLC-MS combination analysis guarantees the purity of a peptide, shown as a peak visible in the HPLC profile, and identity of the desired peptide, determined by ion response of primary amino acid sequence of the peptide.
(II) After Placing Your Order.
- How do I track progress of my peptide order?
Peptide 2.0 provides weekly update on your project, sent to your email. You can also log in your account online to check the status of your order in a real-time. You will receive a notification when your peptides are shipped.
- What is estimated time for my peptide order?
Most of the peptide orders (less than 40 aa) will be finished in 2-4 weeks depending on purity and quantity requirement. For difficult peptide such as hydrophobic peptides, the delivery time will be longer. We will notify you immediately if we could not deliver on time.
(III) After Receiving Your Order.
- What is net weight and peptide content?
Peptides are normally delivered as fluffy lyophilized material. Due to the nature of the peptides, they may still contain traces of moisture and counter ions on protonated amino functions (N-terminus, Arg, His, Lys, etc.). This is not considered an impurity, but reduces the actual peptide content by approximately 10 to 30%. The net peptide weight is the actual weight of the peptide minus the additional weight from residual moisture and counter ions. In order to ensure accurate peptide concentrations, the non-peptide weight needs to be considered and substracted from the gross peptide weight. An accurate determination of the peptide net weight can be performed by quantitative amino acid analysis, which is also available from Peptide 2.0 upon request.
- How do I interpret the data of peptide purity?
With exception of crude peptides requested by customers, which will only be confirmed by MS, all synthetic peptides from Peptide 2.0 will be analyzed by HPLC and MS. HPLC profile will show a peak, representing the main species of mass, thus purity of your peptide (see left panel of Figure below). MS profile shows molecular weight of the synthetic peptide, determined by ion response of primary amino acid sequence of your peptide.
- How do I dissolve my peptides?
The solubility of peptides greatly depends on various parameters like the primary and secondary structure, the nature of modifications made, the solvent, the pH value and the final concentration. Therefore it is almost impossible to predict. If a peptide is hardly soluble in aqueous solution sonification may improve solubility. For basic peptides 10% acetic acid has been recommended whereas for acidic peptides aqueous ammonia or 10% ammonium bicarbonate solution has been shown helpful. Peptides having poor solubility in aqueous solutions organic solvents have to be applied to get these peptides in solution. Use the minimal amount of organic solvent (DMSO is preferred but DMF, DMA, isopropanol, methanol and others can also be used) to dissolve your peptide. We strongly recommend dissolve the peptide first in the organic solvent. In a second step water or other aqueous media should be slowly applied until the final concentration is reached.
- What is the common practice for peptide pools used in ELISPOT or other cellular immune assays?
Peptides are recommended as 15-mer with 11 amino acid overlap (see our peptide generator). Peptides are custom synthesized at 10 mg scale of greater than 90% purity. They can be dissolved in DMSO at high concentration (>10 mg/ml) and stored at -70oC in multiple aliquots. The peptide pools can be made of peptides up to 100 per pool, and are used in T cell assays at concentrations ranging from 1 to 2 mcg/ml for each peptide. Please note, the final DMSO concentration in T cell cultures should be below 2%.
- What are the best storage conditions and how stable are the peptides?
Peptides are normally delivered as fluffy lyophilized material, which are stable and resistant to premature decomposition. Recommended storage condition for lyophilized materials is at -20oC. If peptide is dissolved in solution, it would be best to avoid storing peptides at ambient temperature or 4oC, under which conditions peptides can be oxidized and broken down. The recommended storing conditions are listed below
- Store at -20°C or colder under stable temperature
- Store in small aliquots in solution and avoid repeated freeze-thaw cycles
- Use sterile buffers at slightly acidic pH
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