Frequently Asked Questions
- How do I place my order?
New England Peptide currently offers five options for ordering products:
- Email us at firstname.lastname@example.org
- Call us at 978-630-0020 or 888-343-5974 (toll-free)
- Use our convenient Online Quote System
- Use our convenient Online Catalog Ordering System
- Download an order form and fax it to us at 978-630-0021
- Email us at email@example.com
- How do I check the status of my order?
You may contact our friendly customer service department by email at firstname.lastname@example.org or by phone (888-343-5974) during normal business hours for information on the status of your work.
- What do I do with the ends of my peptides - keep them free or block them?
- Peptides are created to mimic proteins or the cleavage products of proteins. When proteins are cleaved in vivo, they have naturally occurring free unprotected termini. Therefore, blocking the termini is not necessary for in vivo cleavage. However, when the sequence is not a known cleavage product, blocking the termini is necessary in order to mimic the peptide bonds normally found in the parent sequence.
- Use the following rules:
- If the sequence is C-terminal, block the N-terminus by acetylation
- If the sequence is N-terminal, block the C-terminus by amidation
- If the sequence is internal, block both ends with acetylation and amidation
- If my peptide is 95% pure, what is in the other 5%?
Peptide purity is determined by reverse-phase HPLC using a standard gradient established by NEP (1% per minute). During synthesis, the coupling reaction of one amino acid to another is not always 100% efficient, causing a variety of deletion sequences to be generated. Most of the deletion sequences are purified out, but a few may have similar chromatographic characteristics to the target peptide. These remain in the peptide sample and account for the percent of impurities.
- How do I reconstitute my peptide?
- Bring frozen or refrigerated peptides to room temperature in a desiccated chamber to avoid water absorption.
- Always begin by reconstituting a small amount of peptide before committing the entire lot.
- Use sterile water or sterile filtration. If there are any methionine (M), cysteine (C), or tryptophan (W) residues, use oxygen free solvents to prevent oxidation.
- Avoid reconstituting a peptide in a buffer, such as PBS. Salts hinder solubility.
Amino Acid CharacteristicsRecommended SolventHydrophilic residues (KRHDEN)H20Hydrophobic residues (AVLIMFW)Low solubility in aqueous solvents; are soluble in organic solvents (DMF, DMSO, TFA, Acetonitrile)
- Choose the appropriate solvent. Begin reconstituting at a higher concentration than your desired final working concentration.
- A solubilized peptide is completely clear. No flecks or cloudiness should be present.
- If a peptide with more hydrophilic residues is still not completely reconstituted:
- Adjust the pH of the solution according to the overall charge of the peptide.
- Count the possible positive charges (K,R,H and free N-terminus).
- Count the possible negative charges (D,E and free C-terminus).
- Determine which is greater.
- If positive charges are greater, add dilute acid dropwise to protenate residues and maximize charge.
- If negative charges are greater, add dilute base dropwise to deprotenate and maximize charge.
- Try sonication, gentle heat or an organic solvent, such as DMSO, acetonitrile or DMF.
If your peptide is still not completely reconstituted, re-lyophilize the peptide and begin again.
Please contact NEP Technical Service for assistance, should you have further difficulty.
- What is PepTrend™?
PepTrend™ is a software program designed by NEP to facilitate peptide design and synthesis during peptide production. Every sequence is tested in the three areas of peptide production: synthesis, cleavage and purification.
PepTrend™ uses decades of scientifically proven data, along with general trends found during the synthesis of thousands of peptides, to warn of potential problems during any of the three areas of synthesis. The software allows NEP to provide the highest level of service for our customers. NEP can instantly warn customers of potential problems while seeking alternate synthesis strategies before production starts.
- What is net peptide content and what does it mean?
The weight of dry peptide does not consist of peptide only, but includes non-peptide components such as water, absorbed solvents, counter ions and salts. Net peptide content is the actual percent weight of peptide. This number may vary, from 50 to 90 percent depending on the purity, sequence, and method of synthesis and purification. Do not confuse peptide content with purity; they are two distinctly separate things. Purity is usually determined by HPLC and defines the percent of the sample that is the target peptide sequence. Net peptide content only gives information on the percent of peptide versus non-peptide components. Net peptide content is accurately found by performing amino acid analysis or UV spectrophotometry. This information is important when calculating concentrations of peptide during sensitive experiments. If you need help, please ask NEP.
- What purity do I need for my experiments?
The final purity of a peptide is very important and depends on the type of experimentation you are doing:
- For non-sensitive screening assays, crude or >75% is recommended.
- For immunogen grade, >85% is recommended.
- For receptor/ligand studies, bio-assay studies, or cell studies >95% is recommended.
- For structural studies, >98% is recommended.
- How do I store my peptides?
Proper storage of a peptide can prevent bacterial degradation, secondary structure formation, oxidation and other potential degradation for several years. Peptides are most stable in their lyophilized form at -20 deg C or colder in a sealed container containing desiccant.
If peptide must be stored in solution, ensure pH is in the 3-6 range and then aliquot peptide into usable sizes to prevent damage from multiple freeze/thaw cycles. Cysteine (C), methionine (M), tryptophan (W), asparagine (N) and glutamine (Q) are most sensitive to degradation in solution.DurationStorage RecommendationLong-Term Storage3 Months - Several YearsLyophilized powder, frozen and desiccated, -20%uFFFD celsius or coolerMedium-Term Storage0-3 MonthsFrozen liquid (-20 deg celsius) or refrigerated lyophilized powderShort-Term StorageLess than 1 WeekRefrigerated liquid or refrigerated lyophilized powder
- What is the maximum peptide length you can produce?
NEP routinely synthesizes peptides >70 amino acids in length. The longest peptide (protein) we have made synthetically has been 120 amino acids long.
- Why does my KLH/peptide solution appear cloudy?
KLH (Keyhole Limpet Hemocyanin) is a large (MW = 4x105 - 1x107 kDa) aggregating protein. Because of its size and structure, its solubility in water is often limited. This can manifest in tendrils or a general cloudy appearance. This does not affect antigenicity, and the turbid solution can be used for immunizations.
- How can New England Peptide help me with my HTS?
In order to satisfy the growing requirements of the proteomics boom, New England Peptide LLC has designed a Peptide Array program. We have developed a rapid, economical way to synthesize 96 different peptides, unbound, in a 96 individual tube format. Each plate is individually tested for accuracy and can be used for epitope mapping, libraries, protein characterization and much more. We offer up to 15-mer peptides at 2.5 um scale (2-3 mgs of peptide per well). We also offer three tiers of analysis and delivery is only 3 - 4 weeks. Using this format, peptides cost as little as $29 each.
- Which array analysis tier is right for me?
Tier 1 includes a mass-spectral analysis of five statistically significant peptides. This tier is appropriate when the peptide sequences are very similar, sequences are short (seven or eight amino acids), or the research is in a very general or early stage.
Tier 2 includes a mass-spectral analysis of every peptide. We recommend this tier for all of our customers, as it offers the best value and assurance that peptides are correct. Use Tier 2 for all applications, including longer peptides or difficult sequences.
Tier 3 includes a mass-spectral analysis and HPLC profile of every peptide. This tier is useful when HTS is at a later stage and before peptides are singled out for larger scale production.
- Which antibody package is right for me?
Polyclonal antibodies can be unpredictable in their specificity and concentration. If you are doing a quick non-sensitive assay, it may be possible to use the antisera directly without any additional purification. In this case, order our Standard Antibody Package.
If you require a more specific and concentrated antibody (such as for extensive western blotting or staining applications), the Affinity Purified Antibody Package is a more appropriate choice. A good example where affinity purification is helpful is the production of a phospho-specific antibody.
- What species should I choose for my antibody production?
Rabbits are the industry standard as they are highly reliable, affordable, and respond in a reasonable amount of time. Should you need a large volume of serum, goats or sheep are an excellent option. Chickens are ideal for proteins with high homology in mammals. Guinea pigs, hamsters, and rats are great options if you need an antibody to do co-localization work with a rabbit antibody.
- Why do I need two rabbits?
Every animal responds differently to immunization and often animals make different subsets of antibodies to a single antigen. Immunizing two animals increases the odds of one of the animals providing antibodies with high reactivity and specificity to your protein of interest.
- Why should I use a peptide for antibody production?
Anti-peptide antibody production is a useful tool as it allows one to focus on a specific region of a protein. Your resulting antibody may end up more specific, as you can completely regulate exactly what region of the protein the antibody is made against. Using a peptide also increases the chance of successful antibody production, especially on proteins that have an overall hydrophobic trend. Because we can hand select the region that the antibody will be produced against, we are given the freedom to select regions that are particularly antigenic and specific, increasing your chances of a successful project.
- How do you choose the best antigen?
First, we discuss with you what your most important needs are (such as making sure the antibody does not cross-react with protein family members, etc.). We then use that framework to select a recommendation that is antigenic and available within the protein's 3D structure.
- Can I inject more than one peptide into the same set of animals?
Injecting multiple peptides into one set of animals is a useful, cost efficient option that increases your overall chances of success if you only need an antibody to one of the two sequences injected (if they are to the same protein, for example). Often one peptide will elicit a much stronger response than the other, so we cannot guarantee that both peptides will work.
- What chemistry do you typically use for carrier protein conjugation?
We recommend adding a cysteine to one of the termini of your peptide (or designing the peptide to use one that is natural) and using the free sulhydryl group to conjugate to KLH. This is a straightforward, highly efficient and stable chemistry that will not affect the specificity of the resultant antibody.
Several alternative conjugation methods are also available, but only recommended when you have internal or multiple cysteines in your antigen.
- What do I do with my pre-immune serum?
Pre-immune serum is a great negative control for a wide variety of experiments.
- How do you test for reactivity?
The serum from the first production bleed is tested for reactivity against the peptide using an Enzyme-Linked Immuno-Sorbent Assay (ELISA). The peptide is coated onto a 96-well plate, followed by serial dilutions of antiserum that contain the antibody. The measurement of the resultant signal, called a titer, is then sent to you with your shipment.
- How much antibody should I expect to receive?
Antibody production depends on epitope selection, peptide synthesis, carrier conjugation, immunization procedures, and biological systems in animals. Therefore, there is wide variability in the antibody response. The NEP Standard Antibody Package provides ~80mL antisera. The NEP Affinity Purified Antibody Package often yields >10mgs of purified antibody in addition to 40mL antisera.
- How do I store my antisera / antibody?
Sera storage should be at -20° C (for up to one year), or 4° C for less than a month. For longer term storage, add 0.05% azide and freeze. Purified antibody is most stable when stored at -80° C and multiple freeze-thaws are avoided. Alternatively, you can add glycerol to a total concentration of 30-50% and store the antibody at -20° C.
- How long can I expect my rabbits to produce good antibody?
With the inherent variability in polyclonal antibody systems, this is always difficult to predict. As long as the initial response is good, your rabbits should keep producing about the same grade antibody for an average of six months. Many rabbits are productive for longer than one year, but the strength and specificity of your signal can fluctuate during this time. The longer the rabbits are alive the better they acclimate to the antigen, and the response begins to decrease.
- Will I be able to use my antibody in multiple applications?
We actually design the peptide so that it gives our antibodies a very good chance to be able to be used across multiple applications such as western blots, staining, immunoprecipitation, flow cytometry, and ELISAs. Whether it is successful doing so depends on multiple factors, including not only the quality of the antibody that is made but also the specific systems and methods in place that are used for testing. Unfortunately, as these testing systems are entirely out of our control, we cannot make any guarantees that the antibody will work perfectly in the application that you need.
- What are your accreditations?
We only contract with programs that are AAALAC-accredited, OLAW-assured, and USDA-licensed. Use OLAW # A3975-01 if you need to file your project through the NIH.
- What should I use as an antigen?
We can either design and manufacture a specific peptide antigen or use another type of antigen (protein, viral, erc.) that you provide. We will work with you to determine the best antigen for your project needs.
- If I send you material, how much do you need and how should I send it?
5mg is generally enough to cover the needs for both immunizations and screening, but up to 10mg can be required. Material should be sent overnight under similar conditions to how it is stored (dry ice or ice packs).
- What strain(s) of mice should I use?
When immunizing five animals, most customers will use Balb/c mice (Swiss Webster and A/J mice are also regularly available). It is recommended to mix strains when immunizing 10 animals in order to ensure a more varied response.
- What screening method does NEP use?
In the standard monoclonal package, NEP will design a direct ELISA with your antigen bound to a 96-well plate for screening. We can also send hybridoma supernatants to you for testing in your assay. The success of a research project using mAbs often depends on the screening assay used to test the generated material. We will work with you to determine the best screening method.
- What will I receive at the end of the package?
At the end of a successful project you will receive 50mL of supernatant for each of up to two positive sub-cloned hybridomas. Additional material can be purchased through ascites or roller bottle scale-up production.
- What is NEP's policy of ownership and confidentiality?
The customer owns all antibody material produced under contract to NEP. Each order is handled with strict and complete confidentiality.