FAQs

Custom Peptides

With exception of our crude peptides, which will only be checked for identity by MALDI-MS, all synthetic peptides will be analyzed by HPLC and MS. Beside MALDI-MS our routine application involves HPLC-MS techniques to guarantee that a peak visible in the HPLC profile corresponds to the target mass. Since peptides show different ion response depending on the actual primary sequence availability of different MS-equipment is crucial for doubtless analysis. At JPT Peptide Technologies’ state of the art equipment (MALDI-MS, HPLC-(ESI)MS (ion trap and quadrupole) guarantees unambiguous analysis. In addition, JPT Peptide Technologies offers optional amino acid analysis and peptide content determination.

JPT Peptide Technologies offers custom service for peptides ranging from 2 to appr. 150 amino acids. Standard stepwise solid phase procedures provide access to most of the peptides in range from 5 to 50 amino acids in length. For short peptides (i.e. dimers, trimers etc.) and peptides with special modification we have solution phase approaches available. For long peptides up to appr. 150 amino acids we use our proprietary convergent peptide synthesis approach. For longer peptides the success rate drops below 50% and therefore recombinant technologies might be advantageous.

JPT Peptide Technologies offers different purity levels starting with > 70% up to >95%. Ultra pure materials with > 97% purity are available for some peptides upon request. The following list might serve as guideline:

> 70%: immunological applications and nonsensitive screening
> 80%: immunological applications and nonsensitive screening
> 90%: SAR studies, bioassays
> 95%: NMR, crystallization, bioassays
> 97%: NMR, crystallization, sensitive bioassays

JPT Peptide Technologies synthesize custom peptides in a range from one mg (smaller aliquots available) up to several gramms per peptide.

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 will support solubility. For basic peptides 10% acetic acid has been recommended whereas for acidic peptides aqueous ammonia or 10% ammonium bicarbonate solution were shown to be 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 to 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.

JPT Peptide Technologies provides a lot of well established peptide modifications like acetylation, biotinylation, phosphorylation and attachment of fluorescent dyes or quencher pairs but is also specialized in doing unusal reactions. It will be a pleasure to discuss your special needs in detail.

The default ends of a peptide are a free amine group at the N-terminus and a acid group at the C-terminus.
However, often a peptide represents a part of a parental protein sequence. In that case the more “native” ends would relate to blocked N-terminus (i.e. acetyl) and C-terminus (i.e.amide). In addition this modification avoids the introdcution of additional charges and makes the resulting peptide more stable towards enzymatic degradation resulting from exopeptidases.

JPT Peptide Technologies is known for its patented high throughput technologies. Up to hundred thousand isolated peptides can be produced in weeks.

JPT Peptide Technologies possesses a complete range of peptide synthesis techniques including:

• solid phase synthesis and solution phase synthesis techniques
• BOC chemistry and Fmoc chemistry
• stepwise synthesis, convergent synthesis approaches, ligation techniques
• nano scale peptide synthesis to multigram scale
• manual peptide synthesis and automated proprietary high throuput peptide synthesis

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 weigth needs to be considered and substracted from the gross peptide weight. An accurate determination of the peptide net weigth can be performed by quantitative amino acid analysis, which is also available upon request.

Routinely, assembly and purification and quality control of medium size peptides takes 3-4 weeks. In addition JPT Peptide Technologies offers rush order options guaranteeing extremely short delivery times upon request.

All peptides will be shipped lyophilized in small polypropylene vials (2 ml or 10ml) with screw caps. Alternatively, smaller aliquots will be shipped in small amber type glass tubes (1.8ml) with screw caps. All tubes contain labels with information on primary sequence, amount and batch number.
The peptides provided will be accompanied by the original analytical data achieved and an analytical data sheet providing comprehensive information about key characteristics of the peptide such as primary sequence, molecular mass, morphology, counter-ions, purity etc.

Peptides are normally delivered as fluffy lyophilized material, which has been proven to avoid premature decomposition. Recommended storage conditions are:

• Store at -20°C or colder in a dry environment.
• Avoid repeated freeze-thaw cycles.
• Avoid storage of aliquots in solution (Simply order convenient lyophilized aliquots)
• Store at -20°C and use sterile buffers at slightly acidic pH (if storage in solution cannot be avoided)

PepSpots™

JPT Peptide Technologies provides a detailed protocol together with every PepSpot peptide membrane.

In addition to the membrane bound PepSpot peptides ordered JPT Peptide Technologies assembles control peptides on the same membrane which are connected via a cleavable linker system to the cellulose surface. After cleavage of these peptides from the membrane their quality will be checked by MALDI-MS. The control peptides include specific sequences which are difficult to synthesize. The purity of the peptides synthesized varies for each peptide and is dependent on the sequence and length. Our analyses have determined that peptide purity is typically >70% for average 6-15 mers (by HPLC and mass spectroscopy).

Which end of the peptides is coupled to the cellulose membrane?

The peptides are covalently linked to the cellulose support via the C-terminus. For sufficient flexibility for binding studies, a two ß-alanine residue spacer or a short PEG spacer is inserted between the cellulose and each PepSpot peptide.

N-terminal acetylation is recommended for peptide scans. Because peptides are more stable to degradation and the uncharged N-acetyl represents better the region in the native antigen than a charged NH3+-group. There is no extra charge for acetylation.

Yes, as cysteine containing peptides are sensitive to oxidation and cyclization.
Therefore, the substitution of Cys by Ser increases the peptide quality. Secondly, unspecific interactions between cysteine residues and antibodies/proteins could occur which give false-positive results. Finally, Cysteine is very scarcely an essential residue in antibody epitopes.

The PepSpot peptide membranes usually can be used several times. JPT Peptide Technologies provide a protocol including different regeneraton protocols. Only in few cases the regeneration fails due to strong binding of native or denatured antibodies to the peptides or the cellulose membrane.

Detections can be performed with a chemiluminescence substrate in combination with an imaging system or, if not available, with a standard X-ray film and film cassette. Detections via fluorescence read-out are not recommended.

We recommend the use of horseradish peroxidase(HPR)-conjugated antibodies, while other enzymes such as alkaline phosphatase (AP) may also be used. Please note: It has often been observed that AP binds to peptide spots depending on their sequences, whereas HRP in almost all cases did not interact with the peptides. Please also note: Do not use sodium azide as a preservative for buffers with peroxidase as it is an inhibitor of the enzyme.

The spot synthesis technology is compatible with D and L forms of amino acids as well as unnatural amino acids. However, the chemistry requires that the amino acids have an Fmoc protection group at their N-terminus and acid labile protecting groups on their side chains (e.g. Pbf, OtBu, Trt, Boc, tBu, etc). Please note: Some unnatural amino acids may give poor coupling yields due to sterical hindrance.

Yes, JPT Peptide Technologies also offers Cys-Cys-cyclization and biotinylation for PepSpots.

Most linear peptide epitopes for antibody applications are between 3-9 amino acids in length. To be on the save side we recommend a peptide length of 13 amino acids and an offset of 2 to 3 amino acids for the mapping of linear epitopes. Hence, when working with a large protein, it is recommended that the offset does not exceed 3 amino acids in order to localize the epitope. An offset of 1 is recommended for finer mapping of the epitope.
For the mapping of conformation dependend (discontinuous) epitopes we recommend a peptide length of 15 to 20 amino acids and an offset 5 amino acids in maximum.

The amount of peptide that is synthesized is determined theoretically from calculating the nmoles of free amines available for coupling per Spot and assuming a coupling efficiency of >98% per cycle. A typical synthesis is expected to yield between 5-10 nmol (6-12 µg for an average 10mer peptide).

The PepSpot peptides have a diameter of approx. 2-3 mm.

The peptides are synthesized in a grid fashion with 0.37 cm x 0.37 cm (0.15 in x 0.15 in, center-to-center) spacing, 20 peptides per row. Other formats are available upon request.

JPT Peptide Technologies offers two types of PepSpot peptide membranes:

PepSpots:
The connection between the peptide and the membrane is an ester bond and therefore labile towards strong bases (i.e. NaOH) and nucleophiles (i.e. NH2R). The peptides themselves are quite stable. However, strong acids may occasionally break acid labile amide bonds such as Asp-Pro. In addition, there are several amino acids such as Cys or Met which are sensitive towards oxydation. Finally, very strong bases (NaOH) may hydrolize peptide bonds as well.

PepSpots (PEG):
The connection between the peptide and the membrane is an ether bond and therefore stabile towards strong bases (i.e. NaOH) and nucleophiles (i.e. NH2R). We suggest to use this membrane type if electroblotting experiments or application of alkaline phosphatase is planned.

The membrane material is stable to the synthesis conditions, but should not be exposed to strong acids for prolonged periods.

New membranes should be stored at -20°C until use. Incubated membranes which will be used again after only a few days should be kept with a small volume of T-TBS buffer in a Petri dish at 4°C. Incubated membranes which will be stored for a longer period should be regenerated and kept at -20°C.