Frequently Asked Questions

Here you can find answers to the questions most frequently asked by our customers. You can directly go to a question by clicking on it in the table of contents.

In case you cannot find the answer to your question, you are cordially invited to direct your inquiry to us. You can further contact our local representatives or one of our distributors in China, Europe, Korea, Japan, Jordan or the United States.

 

Get answers by clicking on a question:

 

 Technical questions


 

Technical questions

What distinguishes the PEPperPRINT technology from conventional peptide microarray technologies?


Our technology is based on a patented peptide laser printing technologywhere peptides are directly synthesized onto glass slide. For epitope mappings, we translate proteins into 15mer peptides with an overlap of 14 amino acids (corresponds to a shift of 1). This leads to a maximum peptide peptide overlap and enables a high resolution epitope analysis. A reduction of the peptide peptide overlap results in an inaccurate outcome of epitope lengths and is therefore not recommended.

 

Since we synthesize the arrays directly onto glass slides, we are able to design each array individually and we are flexible in the selection of custom peptide sequences. At PEPperPRINT you do not pay per peptide, but per array and, if desired, service. Other providers charge each synthesized peptide. Their peptides are costly pre-synthesized in high amounts and afterwards spotted onto the glass slides. Due to the fact that this pre-synthesis is expensive, other providers offer a lower peptide overlap (correlating with a higher shift) to reduce the number of peptides they have to synthesize.

 

Our service includes comprehensive data analysis, including epitope prediction and interpretation. A report tailored to your specific project and needs includes all experimental data, images and discussion of the obtained results.

You can find additional information on amino acid microparticles and glass slide coatings on our webpage. Moreover you can read testimonials by some of our customers.

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How many amino acids can be coupled with the PEPperPRINT technique? What is the maximum length of peptides that you can print on a custom chip?


We routinelysynthesize 15mer peptides. In an optimal setting we could demonstrate a successful synthesis of 25mer peptides: We synthesized an elongated (GS)n linker up to amino acid 17, and added the Flag epitope, consisting of 8 amino acids, on top. We could show α-Flag antibody binding to these 25mer peptides, meaning that the peptides were synthesized up to amino acid position 25.


Routinely we print 15mer peptides, but we can also provide shorter or longer peptides, up to 20 amino acids in length. For peptides between 17-20 amino acids we need to charge an extra fee per chip.

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Which peptide length/overlap do you use?


For our epitope mapping services we routinely use 15mer peptides and the maximum peptide-peptide overlap of 14 amino acids (equals to a shift of one). If desired, we can also adjust the peptide lenths and/or the overlap.

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How long does it take to print one custom peptide array?


Array production takes about 3-4 weeks including quality control steps.


Details about the process of peptide array production can be found here.

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What is the spot size and amount of peptide per spot?


The spot size is approximately 150x300 µm and the distance between spots is 254 µm (horizontal) and 508 µm (vertical). The peptide concentration per spot is in the lower picomolar range.

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How many array copies can be printed per slide?


We can print 1, 2, 3, 4, 5, 8 or 16 array copies per chip, which can be assayed individually with a matching incubation tray. The number of array copies per chip depends on the number of peptides per array.

  • 1 array per chip with up to 4,290different peptides in duplicate
  • 2 array copies per chip with up to 1,914differentpeptides in duplicate
  • 3 array copies per chip with up to 1,056 differentpeptides in duplicate
  • 4 array copies per chip with up to 726 differentpeptides in duplicate
  • 5 array copies per chip with up to 594 differentpeptides in duplicate
  • 16 array copies per chip with up to 100 different peptides in duplicate


In addition to these peptides, we furthermore frame each array with HA control peptides.

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How are the spot duplicates arranged?


Due to the printing flexibility, spot duplicates can be arranged randomly, beneath or next to each other.

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How are the peptides coupled to the glass slide?


We use a linker consisting of 3 amino acids (2x β-alanine and 1x aspartic acid). The aspartic acid is negatively charged and prevents the unspecific binding of the negatively charged fluorescent dyes to positively charged lysines close to the array surface.

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Does the technique work equally well for hydrophilic and hydrophobic epitopes and how sensitive is the assay?


Our assay conditions are adapted to detect epitopes with different degrees of hydrophobicity/hydrophilicity. Even weak epitopes with sub-micromolar affinity can be detected.

We assessed the assay sensitivity in an own study. We synthesized Leu-enkephalin peptides and variants on the array and incubated them with an α-Endorphin antibody. We then compared the fluorescent signal intensities with published apparent affinities. We were able to detect antibody binding down to µM binding affinities. Since µM-binding affinities are ranked as weak affinities, we can conclude that our assay sensitivity is very high. For further information, please have a look at this application note.

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How do I know if my antibody binds to linear or conformational epitopes?


We recommend testing your antibodies in Western blot under denaturing conditions. If the antibodies work under these conditions, they likely recognize a linear epitope, which we can map. If you cannot detect bindings in Western blot your antibody might bind to conformational or discontinuous epitopes. To analyze antibodies binding to certain 3D structures, we can print cyclic constrained peptides.

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Can linear and conformational epitopes be detected?


Linear epitopes can be mapped with linear peptides on chip (epitope mapping and epitope substitution scan). For conformational epitope mapping we offer cyclic peptide microarrays that mimic the 3D structure of the epitope.

Detailed information on conformational epitope mapping can also be found in this application note.

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Which types of amino acids can you couple to the chip beyond the 20 standard amino acids?


Beyond the 20 standard amino acids, we offer a variety of different amino acids that can be coupled to the chip. These are:

  • citrulline
  • ornithine
  • D-alanine
  • D-aspartic acid
  • D-cysteine
  • D-glutamic acid
  • D-lysine
  • D-tryptophan
  • D-valine

More D-amino acids are available on request.

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How stable are the printed arrays? How long can they be stored?


PEPperCHIP® Peptide Microarrays should be stored at 4°C and in the dark to ensure a long shelf life. If handled with care, chips are stable for months. If possible, please store them under inert gas atmosphere.

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Can arrays be used several times?


In theory the arrays can be stripped. However, the stripping efficiency always depends on the nature of the samples and the assays. It may not work in all cases, and the chips may also degrade to some extent. Stripping efficiency has to be checked by microarray scanning, and even then you likely will not know if you removed all primary antibodies, or only the secondary antibody. Since we can print multiple array copies per chip (number of copies depends on the size of the array) we recommend using a new array for each experiment.

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Quality assessment and controls

How do you guarantee the quality of your products?


PEPperPRINT is ISO 9001 certified. You can find a summary of all quality control steps here. For further questions please contact our quality control manager.

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How is the accuracy of the (printed) peptide sequence checked?


Due to the high number of different peptides (up to 200,000 on a whole plate) and the picomolar mass, it is technically impossible to analyze all synthesized peptides in HPLC. Nevertheless, we perform numerous quality tests during production setup. We routinely run double coupling steps to get as close as possible to quantitative coupling yields. After coupling, non-reacted amino groups are capped to avoid the formation of peptides with incomplete sequences. According to HPLC analysis and mass spectrometry with reference peptides, side reactions like aspartimide and β-lactam formation or racemization can be excluded.

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How do you reduce errors by interaction between slide surface and peptide?


We synthesize the peptides from C- to N-terminus on chip. We routinely run double coupling steps to get as close as possible to quantitative coupling yields. Moreover we have a custom polymer coating between the glass slide and the peptides that (1) provides a high loading of functional groups and thus peptides, (2) is equipped with long poly(ethylene glycol) linkers to ensure proper peptide accessibility in a hydrophilic environment and (3) blocks non-specific background interactions with proteins and sera.

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How is unspecific binding minimized?


We have developed an own glass slide coating that is optimized for peptide array production and antibody assays, and offers superior signal-to-noise ratios. The arrays are blocked prior to antibody hybridization with either BSA or blocking reagents (like Rockland Blocking Buffer) to prevent unspecific antibody binding.

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How selective is the immunostaining procedure?


This highly depends on the specificity of the target antibody. To prevent non-specific binding, the target antibody staining (with first antibody or serum) is performed over night at 4°C.

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Which control peptides do you use?


We routinely use HA control peptides (YPYDVPDYAG), and our standard arrays include Polio, HA and c-Myc controlpeptides. Other controls are available upon request.

 

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PEPperCHIP®: Requirements, handling and analysis

How do I design my peptide array most efficiently?


We support you with your optimal array design and project setup. Please contact us to discuss your requirements.

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What do I need to know about PEPperCHIP® handling?


PEPperCHIP® handling and analysis is straightforward and follows standard immunoassay protocols. To get a first glance about working with PEPperCHIP® Peptide Microarrays, we recommend watching our short tutorial movie. More details are available in our standard immunoassay protocol. Typical lab and material requirements for PEPperCHIP® Peptide Microarray assays are listed below.

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How do I analyze my peptide microarray?


PEPperCHIP® Peptide Microarray analysis can be done in your own lab or by PEPperPRINT experts within a PEPperMAP® Service Offer. Further information on this can also be found in the corresponding section in this FAQ document.

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What equipment do I need for array staining and analysis?


Below you can find a list of material that is needed for peptide array experiments. PEPperPRINT offers different material for peptide array experiments (full descriptions can also be found here).


 

Material supplied by you (if you decide to perform experiments yourself):


  • Orbital shaker
  • Microarray Scanner: The PEPperCHIP® Peptide Microarrays are compatible with most microarray scanners (e.g. GenePix 4000B, 4100B and 4300/4400 Microarray Scanners, Tecan Laser and Power Scanner, Agilent High-Resolution and SureScan Microarray Scanner, Innopsys InnoScan 710 and 900, NimbleGen MS 200 Microarray Scanner, LI-COR Odyssey Imaging System and many more). A detailed scanner list can be downloaded here.
  • Buffers: PBS, Tween
  • Blocking reagent: BSA or Rockland Blocking Buffer
  • Labeled secondary antibody for staining of primary sample (e.g. mouse α-human antibody). Please note that strongly charged fluorescence dyes can interact with acidic and basic side chains of peptides. We thus recommend the use of neutral dyes or dyes with few charged functional groups (e.g. DyLight680, DyLight800, Cy3, Cy5), as well as pre-incubation with secondary antibodies to screen for background interactions. PEPperPRINT uses Pierce infrared dyes and the LI-COR Odyssey® Infrared Imaging System.

You can also watch our short tutorial to get an impression about chip handling or download our immunoassay protocol.

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Which scanners are compatible with the PEPperCHIP® Peptide Microarrays?


All scanners that are compatible with 75mm x 25mm (3in x 1in) microscope slides can be used to scan PEPperCHIP® Peptide Microarrays. We recommend a scanning resolution of 20 μm or higher (10 μm, 5 μm, etc.).

Please download our detailed list of compatible scanners here.

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Which fluorescent labels work with the PEPperCHIP® Peptide Microarrays?


Strongly charged fluorescence dyes can interact with acidic and basic side chains of peptides. We thus recommend the use of neutral dyes or dyes with few charged functional groups (e.g. DyLight680, DyLight800, Cy3, Cy5), as well as pre-incubation with secondary antibodies to screen for background interactions. PEPperPRINT uses Pierce infrared dyes and the LI-COR Odyssey® Infrared Imaging System.

 

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PEPperMAP® Service

What does the PEPperMAP® Service include?


During epitope mapping, the peptide array is first incubated with the unlabeled primary antibody. Afterwards, the secondary, dye-labeled antibody is added, which binds to the primary antibody.


Our service includes comprehensive data analysis, including epitope prediction and interpretation. A report tailored to your specific project and needs includes all experimental data, images and discussion of the obtained results. Considering that every customer project is different we adapt our reports to each project. We provide you with all raw data (Excel format) and a report including the following data:


-          Information about the project

-          Assay conditions

-          Array scans

-          Analysis of the epitope

-          Interpretation of the data

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Which epitope mapping options do you offer?


We offer different PEPperMAP® Services:

The PEPperMAP® Assay Service includes all immunoassays with your antibody sample, comprising pre-stainings, incubation with your antibody sample as well as stainings with control and secondary antibodies. Additionally, we quantify and evaluate your data and provide you with a scientific report and the raw data.

This is the most straightforward way to identify antibody epitopes of mono- or polyclonal samples.


With our PEPperMAP® Single or Dual Isotype Assay Service, we perform all immunoassays with your serum samples, pre-stainings and control and secondary antibodies. The PEPperMAP® Single Isotype Assay Service comprises data quantification and analysis of one Ig isotype (e.g. IgG or IgM), whereas the PEPperMAP® Dual Isotype Assay Service comprises the same for 2 Ig isotypes.


With our PEPperMAP® Epitope Substitution Scan the influence of each amino acid of the epitope on antibody binding can be determined. We exchange all amino acids of the epitope with all other remaining 19 amino acids and incubate the generated peptide microarray with your antibody sample. After data quantification and analysis, we provide you with a scientific report.

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I already know my epitope sequence. Can you help me in determining the amino acids that are important for binding?


In case the epitope sequence is known, we are able to identify the amino acids that are important for the binding of the antibodyby a substitutions scan. In this process, each amino acid of the epitope is replaced one after the other by all remaining 19 L-amino acids (D-amino acids on request). The number of peptides can be calculated by


length of the epitope x number of different L-amino acids x number of replicates per peptide


For example, a peptide microarray for an epitope with 13 amino acids contains 13 x 20 x 2 = 520 peptides.


Examples for this service can be found here.


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What are the prices  for the PEPperMAP® service?


PEPperMAP® Service costs are calculated based on the time and material needed for thorough service. Standard PEPperMAP® epitope mapping services with an antibody or serum sample against a single protein are covered by flat-rate fees that are widely independent of the length of the protein sequence. Please do not hesitate to contact us for more information and nonbinding cost estimations for the intended research project. We guarantee to always provide you with the most cost effective PEPperMAP® service solutions available.

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What is the timeline of the PEPperMAP® Service?


The timeline starts with receipt of the antigen sequence, followed by 4 weeks for peptide microarray generation. We usually ask to ship the sample(s) before peptide microarray finalization. Subsequently, the assays will take around 3 weeks followed by another week for data evaluation and quantification. In total, it takes around 8 weeks for the full process until delivery of the report.

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What is the procedure of the PEPperMAP® Service?


For service to start, we need a purchase order containing the order number and the billing address. Furthermore, we need the peptide list/antigen sequence (FASTA format or Uniprot ID).


We will then send you an order confirmation and the layout of the microarray (peptide map) for your approval. After your approval, we will start with the peptide synthesis which will take about 3-4 weeks.


During this time, you can send the samples to the address below. Please inform us as soon as you send the samples and complete the PEPperMAP® Sample Submission Form (containing information about the sample name, storage temperature etc.).


Payment can be done via bank transfer (after receiving the invoice) or by credit card.


More details can be found in this infographic.

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What is the address to send the antibodies to?


PEPperPRINT GmbH
Rischerstr. 12
69123 Heidelberg
Germany

Contact person: Gregor Jainta, Gregor.jainta(at)pepperprint.com, +49 (0)6221 7264489

Please complete and return the PEPperMAP® Sample Submission Form along with your antibody and serum sample(s) to ensure proper handling and storage.

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Are there any special requirements for shipment of samples?


Most customers send us antibody or serum samples on ice packs using FedEx. If you would like to use dry ice, fill the box half with dry ice. Seal your tubes with parafilm, put them in a plastic bag and put this bag directly in the dry ice. Fill the empty space with Styrofoam to prevent that the dry ice is shaken too much.


Please describe your goods precisely on the Air Waybill (e.g. human monoclonal antibody, murine serum, synthetic antibody). If you use e.g. “reagent for research” or “protein solution”, FedEx will ask us for more information, which can delay sample shipment.


For analysis of animal-derived antibodies and sera that are shipped from overseas (e.g. from USA), please complete and return the PEPperMAP® Sample Declaration Form along with your samples to avoid any clearance delay. Animal derived samples are randomly checked by the veterinary office at the airport in Cologne. The veterinary office wants to make sure that no animal diseases or pathogenic agents are introduced into Germany. An inspection by the veterinary office may lead to extra costs. To import animal derived samples into Germany, it is necessary that you state in the PEPperMAP® Sample Declaration Form that your samples have been separated from plasma and are highly purified, so that animal disease or other pathogenic agents were killed off or effectively removed. Please insert the sample nature and state how the samples were produced. Please add this form to your shipment documents outside of the box.


If you have animal serum samples that are not purified, please contact us for further details.


Human serum samples and synthetic antibodies do not pass the veterinary office and thus do not require the abovementionedform.

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What do I need to provide for an epitope mapping?


First, we need the antigen sequence in FASTA format or the Uniprot ID. We will discuss with you how much serum or purified antibody we need to run the assay services for your specific project.

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What sample quantity/concentration is required for an experiment?


The amount of sample needed depends on different factors. One is the incubation volume that depends on the size of the array. Moreover, it depends on whether serum or antibody is used. Last but not least, we test up to three different dilutions for incubation in order to obtain the best results possible. The exact amount of sample needed can therefore only be determined individually for each project.

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Are there special requirements for the protein labeling service?


Please send us your protein sample/antibody without BSA and without sodium azide. Both additives reduce the labeling efficiency.

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Do you test non-specific bindings of secondary antibodies?


Pre-staining with the secondary antibody is a routine background control.

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