Here we provide a selection of frequently asked questions about our most common catalog formats.
1. What is a PepMix?
PepMixes are peptide pools generated by chemical synthesis and pooling of individual 15meric peptides overlapping by 11 amino acids, spanning entire protein antigens or of selected MHC class-I or class-II-restricted epitopes.
2. How much peptide is in a vial?
Each vial contains 25ug (or 15 nmol) of each of the individual peptides. However, in some cases we also offer larger amoun(200µg, or 120 nmol, of each peptide). Please refer to the product datasheet provided on the product page.
3. How many peptides are in a mix?
Depending on the length of an antigen, a mix can contain between 10 and several hundred peptides.
4. How are PepMix delivered?
They are delivered freeze dried at room temperature.
5. How do I store my PepMix?
Freeze dried PepMix should be stored at -20°C or lower for long term storage.
6. How do you ensure that the pool does not contain byproducts that cause inhibition of T-cell responses or give false positive responses?
JPT developed synthesis and purification protocols that avoid presence of contaminants causing false positive T-cell responses or inhibition of T-cell responses by toxic byproducts. In addition, JPT established QC procedures ensuring a high level of batch to batch reproducibility.
7. How do I reconstitute my PepMix?
The best solvent to dissolve a PepMix is DMSO, followed by slow dilution with an appropriate buffer system. Please note that dissolved peptides have a limited stability. Therefore, we recommend immediate freezing of aliquots that you will not use shortly after dissolution. JPT also offers freeze dried pool aliquots.
8. What is the maximum concentration of DSMO I can use in my T-cell Assay?
Based on literature, we recommend a maximum DMSO concentration below 1% (v/v) in cellular assays.
9. What peptide concentration do you recommend for stimulation?
The recommended concentration for stimulating PBMC's is 1 µg/ml of each individual peptide.
10. Can I stimulate whole blood samples?
Successful stimulation of whole blood samples has been described. However, the optimal peptide concentration may be significantly higher compared to PBMC stimulation.
11. What can I do if my protein/antigen of interest is not available as a PepMix?
Simply send us your antigen sequence or Swiss Prot accession, Uniprot identifier or sequence from other protein databases to receive a quote on your customized PepMix. Contact: firstname.lastname@example.org.
12. Are you providing individual peptides and matrix pools for T-cell epitope identification?
Yes, please contact our customer support: email@example.com
13. Are you offering custom pooling and aliquotation service?
Yes, we recommend to purchase freeze dried pools and single peptide aliquots for enhanced stability. Please contact our customer support for advice on the best format. Our pooling and aliquotation protocols are validated and certified according to DIN ISO 9001:2015. We guarantee presence of all peptides, high accuracy of aliquotation and batch-to-batch reproducibility.
14. What pools do you recommend for standardization or positive controls for T-cell assay such as ELISA and CFC?
JPT's CEFX Ultra SuperStim Pool is a Positive Control Pool of 176 known peptide epitopes for a broad range of HLA sub-types and different infectious agents for T-cell stimulation of populations with a diverse ethnic background. This positive control PepMix has been shown to be able to stimulate CD4+ and CD8+ T cells simultaneously. We also offer CEFX Ultra SuperStim MHCI and MHCII subsets for more specific stimulations. For efficient in vitro stimulation of antigen-specific CD4+ and CD8+ T cells, we also have a broad range of antigen specific PepMixes such as HCMV (pp65 , -IE-1 and IE-2) and others .
1. How are PepStar microarrays shipped?
The slides are shipped at room temperature.
2. How should they be stored?
For long-term storage JPT recommends storage at 4°C. JPT recommends NOT freezing the microarrays since the effects of freeze thaw cycles on the glass surface are unclear. Properly stored, our peptide microarrays are stable for 6 months.
3. What is the chemical stability of the microarrays/immobilized peptides?
As long as the microarrays are kept dry and cool (+4°C), the microarrays are stable for 6 months from date of delivery. The bond between peptides and microarray surface is stable in a range of pH 4 -10. Higher or lower pH values might lead to signal loss due to degradation.
4. What is the concentration of each peptide on the microarrays?
The amount of peptide immobilized in one spot (100µm diameter) depends on the surface loading of the peptide microarray. Experiments showed a loading capacity of 4pmol/mm³. Therefore, the amount of peptide immobilized in one spot is approx. 30 fmol/ peptide.
5. What is the size of peptide microarrays?
JPT´s peptide microarrays are compatible with most scanner system accepting microarrays of 1 x 3 inches (or 2.5 x 7.5 cm).
6. What is the layout?
All our microarrays display each peptide in several copies to ensure verifiable results. The exact layout for each microarray is noted in the product description and datasheet.
7. Can the microarrays be used again? Can the microarray be regenerated?
JPT recommends using the arrays only once. Stripping bound proteins or antibodies from the surface can damage it. Plus, very low remainders of protein or antibody may cause a very high background.
8. What kind of hardware do I need for the readout of my peptide microarrays?
a. For low density peptide microarrays (up to 3 x 384 Peptides/Microarray):
- Minimum scanning-resolution: 50 um / Pixel - Focusing should be possible
- Intensity should be adjustable -Size of slide: 1 x 3 inch
- Size of scanned area should be at least 63 x 21mm
- Extinction/emission filters depending on the fluorophores used
b. For high density peptide microarrays (more than 3 x 384 Peptides/Microarray):
- Minimum scanning resolution: 10 um / Pixel
- Focusing should be possible - Intensity should be adjustable - Size of slide: 1 x 3 inch
- Size of scanned area should be at least 63 x 21mm
- Extinction/emission filters depending on the fluorophores used
- Software capable of reading and analyzing .gal-files.
c. JPT is working with a Genepix Axon 4000B and a Genepix 4300 Scanner system. The resulting image should be an 8- or 16-bit format to be compatible with most microarray processing softwares.
9. How does JPT quality control peptide microarrays?
With each synthesis batch we run well selected control peptides that are analyzed by HPLC-MS and need to pass quality control. These control peptides include sequences that are difficult to synthesize. After printing, we scan each microarray and check for completeness and uniformity. In addition, process control peptides (Flag-tag) are included in each synthesis, allowing an independent assay control by application of appropriate antibody combinations.
10. How are peptides immobilized onto the microarrays?
Peptides are covalently linked to the microarray surface chemoselectively via the N-terminus. A flexible linker is inserted between peptide and microarray surface ensuring availability of the binding sites within the peptide.
11. Do you recommend substitution of cysteine residues by serine residues?
Yes, as cysteine containing peptides are very sensitive to oxidation and cyclization, substitution of Cys by Ser increases peptide stability. In addition, unspecific interactions between cysteine residues and antibodies/proteins sometimes occur and give false-positive results.
1. How does JPT Peptide Technologies QC the synthesis of membrane-bound peptides?
In addition to the membrane bound PepSpot peptides ordered, JPT Peptide Technologies assembles control peptides on the same membrane, that 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 ~50% for average 6-15 mers (by HPLC and mass spectroscopy).
2. Which end of the peptide 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.
3. When do you recommend acetylation of the N-terminus?
N-terminal acetylation is recommended for peptide scans, because peptides are more stable to degradation and the uncharged N-acetyl better represents the region in the native antigen than a charged NH3+-group. There is no extra fee for acetylation.
4. Do you recommend substituting cysteine with serine residues?
a. 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.
5. How are the data from the PepSpot incubation evaluated?
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.
6. Which enzyme-labeled antibodies are recommended for detection?
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, leading to unspecific signals, 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.
7. Can unnatural or modified amino acids be used in the synthesis?
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.
8. Can the attached peptides be cyclized or biotinylated?
Yes, JPT Peptide Technologies also offers Cys-Cys-cyclization and biotinylation for PepSpots.
9. What length of offset do I need for peptide scans?
Most linear peptide epitopes for antibody applications are between 3-9 amino acids in length. To be on the safe side, we recommend a peptide length of 13 to 15 amino acids and an offset of 2 to 4 amino acids for the mapping of linear epitopes. Hence, when working with a large protein, it is recommended that the offset does not exceed 4 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 dependent (discontinuous) epitopes, we recommend a peptide length of 15 to 20 amino acids and an offset 5 amino acids in maximum.
10. How much peptide is synthesized per spot?
The amount of peptide that is synthesized is determined theoretically from calculating the amount of free amines available for coupling per Spot and assuming a coupling efficiency of >98% per cycle.
11. What is the size of a PepSpots peptide?
The PepSpots peptides have a diameter of approx. 2-3 mm.
12. What is the density of peptides on the membrane?
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.
13. What is the chemical stability of the membrane?
The membrane material is stable to synthesis conditions, but should not be exposed to strong acids for prolonged periods.
14. What are the appropriate storage conditions for the membrane?
New membranes should be stored at -20°C until use. Incubated membranes which will be reused within several 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.
15. Do you typically put only a single ß-Ala between the target peptide and the linkage to the membrane, or is this a short / poly-ß-Ala sequence?
a. The linker system consists of 2 ß-Alanine residue.
16. What are the PEG-linker options (e.g. PEG6 or PEG10?)
The PEG linker is fixed on a Trioxa residue + 1 ß-Ala. There are no other PEG-options available.
17. What would be the maximum number of peptides that can fit on a 120 x 75 mm membrane?
The spot size is approx. 2mm with a distance of 3.7mm from spot to spot. So 29 peptide plus controls will easily fit into a membrane this size. The maximum number of peptides would be approx. 608 peptides (32x19 spots).
18. Do you provide a grid or map of the peptides that are arrayed?
A sequence list is provided.
19. Are the spots outlined in any way? How do we know where to punch out?
a. Peptides rows and columns are outlined on the membrane. Peptide spots could also be easily identified using UV-light.
1. Which types of SpikeTides are available?
- SpikeTides: light proteotypic peptides with C-terminal Lys or Arg residue
- SpikeTides TQ: light fully quantified proteotypic peptides with C-terminal Lys or Arg residue. Each peptide carries an additional C-terminal tryptic tag, which needs to undergo tryptic digestion to release the respective proteotypic peptide.
- SpikeTides L: heavy labeled peptides with C-terminal Lys or Arg residue
- SpikeTides TQL: heavy labeled fully quantified proteotypic peptides with C-terminal Lys or Arg residue. Each peptide carries an additional c-terminal tryptic tag, which needs to undergo tryptic digestion to release the respective proteotypic peptide.
- Maxi SpikeTides Q-AAA: light fully quantified proteotypic peptides with C-terminal Lys or Arg residue with high purity. Each peptide is quantified by AAA.
- Maxi SpikeTides QL-AAA: heavy labeled fully quantified proteotypic peptides with C-terminal Lys or Arg residue with high purity. Each peptide is quantified by AAA
- SpikeTides TQL Plus: heavy labeled fully quantified proteotypic peptides with C-terminal Lys or Arg residue. Each peptide carries an additional C-terminal tryptic tag, which needs to undergo tryptic digestion to release the respective proteotypic peptide. Peptides are purified (>95% purity) and provided in aliquots of 10 nmol to enable enduser-based re-quantification.
2. Why is the tag necessary?
For SpikeTides TQ and SpikeTides TQL, the tag carries a moiety enabling the quantification of the target peptide during HPLC-runs using specific wavelengths. The tag is optimized to enable efficient cleavage using standard proteases like trypsin. After cleavage, the proteotypic peptide with the native C-terminus is available for the MRM assay.
3. Are the termini of the peptides modified in any way?
As necessary for proteotypic peptides, the N-termini are free, the C-termini are acidic.
4. What amount of peptide is in each well?
For the SpikeTides, a rough estimation of the peptide amount is given in the product documentation. The estimatated peptide amount is based on individual quantification (based on UV-absorption and amino acid composition of target peptide) of random samples from customer library. For the quantified SpikeTides TQ and SpikeTides TQL, the synthesized peptides are aliquoted in 5x1nmol fractions. Quantification is based on HPLC-MS analytics and UV read-out of the Quantification-tag.
5. How much enzyme is needed for the cleavage of the tagged SpikeTides?
The cleavage of the SpikeTides can be performed in the same assay solution as the tryptic digest of the target protein takes place.
6. How do I know whether the cleavage was successful?
JPT Peptide Technologies has validated the cleavage efficiency in many conditions. Results are available here . The efficiency of the cleavage can be checked by doing HPLC-runs after the cleavage was performed. The signal of the cleaved tag should be visible at a retention time of 0 to 0.5min (C18, 3µ,, 20x2mm, 1ml/min flowrate, 6 minute gradient, 5% to 95% Acetonitril water) and should light up when analyzed at 350nm.
7. What is the chemical stability of the peptides?
For long-term storage JPT Peptide Technologies recommends to keep the plates / racks sealed below 4°C, ideally, for highest stability at -80°C. We recommend using the freeze dried peptides up to 6 months after delivery of the peptides. Once thawed and dissolved in buffer solution, JPT Peptide Technologies recommends to perform the assay without further delay to avoid degradation of the peptides.
8. How are the plates delivered?
The plates or micronic racks are shipped at room temperature. For long-term storage JPT Peptide Technologies recommends to keep the plates / racks sealed below 4°C, ideally, for highest stability at -80°C.
9. Do I need special reagents/chemicals?
For SpikeTides TQ/TQL peptides, a sample of trypsin or another suitable protease is required to cleave the tag. Potentially, other required chemicals are described in the protocol and are dependent on the modifications of the library ordered. SpikeTides peptides don't have an additional Q-tag at the C-term and can be used directly without prior tryptic digestion.