New England Peptide

Our custom polyclonal antibodies are typically robust and specific, but there are times when the very nature of their varied response can limit your research. When you need a long term solution to obtaining a homogenous supply of reliable and continuously reproducible material, a monoclonal antibody is where you turn.

Whether you wish to use peptides, proteins, or something entirely novel, we will communicate with you to develop the right strategy to meet your needs.

Below is our standard service broken down into the typical multi-phase development plan. We can easily customize various aspects of this to your specifications whenever necessary. We produce anti-peptide monoclonal antibodies as well as anti-protein monoclonal antibodies.

Phase I: Inception

• NEP will analyze target protein and suggest peptide antigen, or customer can supply material for immunization
  
• Proper screening methods will be discussed and finalized
  
• Four mice (Balb/c) will be immunized over a period of seven weeks
  
• Anti-sera will be analyzed by ELISA using appropriate material and/or samples sent to customer for evaluation
  

Phase II: Development

• Splenocyte cells from top two mice will be fused with a myeloma cell line
  
• Fusions will be screened for hybridomas producing specific antibodies by ELISA
  
• Expanded fusion positives will be screened in a reconfirmation assay from which up to twenty different parental clones will be selected and supernatant samples sent to customer for evaluation
  
• One vial of each parental clone will be frozen and stored for future use
  

Phase III: Production

• Sub-cloning of 1-2 parental cell lines with ELISA screening and expansion
  
• Up to two positive subclones from each parental clone will be expanded and isotyped, with a final deliverable of 50mL supernatant from each sent to the customer for evaluation
  
• All remaining material is frozen and can be banked for a period of one year
  

Some additional available options are:

• Scale up and purification
  
• Additional samples (>20) of clone supernatants
  
• Additional sub-clones

It’s been our mantra from the beginning, and this philosophy will never change at NEP. No matter what type of project you have or what type of antigen you wish to use (peptide or protein), you can trust us with your antibody development. We do everything possible to make certain you have the results you expect and deserve, and our animal facilities are AAALAC accredited, OLAW assured, and USDA licensed to ensure all proper care is taken and research animals are treated appropriately.

If you are frustrated by the quality of anti-peptide catalog antibodies you receive, or are working with a novel target unavailable in catalogs, custom antibody production - with the flexibility to design a peptide antigen catered to your own needs - is the logical place to turn. As a core competency, making clean and pure peptides has always been something that NEP has done well. However, even if using the purest peptide possible, unless that peptide is designed the right way you may not get the results you are looking for. Over the past 15 years, we have developed a proprietary process for this design that works. This comprises quite a bit more time and effort than our competitors are willing to put in, but the results - happy customers - are worth it.

Our Design Philosophy

First and foremost we know it is important to listen to your needs and react accordingly. More often than not, however, the design results that exemplify a quality peptide for antibody production will follow these three main tenets:

• Specificity – We ensure the antigen has a low chance of cross-reacting with closely related family members or proteins from a similar class.
  
• Antigenicity – We ensure the antigen will likely produce a strong, measurable response. Our average ELISA titer across all our varied projects is >1:1,000,000.
  
• Structure – We design our sequences to mimic the normal protein structure as best as possible in order to generate antibodies that have the best chance to bind to the protein (not only the peptide) across multiple assay formats.
  

There are times where one of these aspects will be in conflict with another. Rest assured that in these cases we work with you to find the best compromise to fit your needs.

The design process (included in all projects) is only one of the benefits you gain from working with us. Our dedication to fast, helpful service is another. Whether you have a project with layers of special design and in-process work, or you have a purified protein you want to send us for the generation of anti-sera, we treat you like you are the only customer we have.

Our Standard Rabbit Polyclonal Antibody Package - Anti-Peptide Antibodies

Rather than itemize every last detail, we keep it simple with all-inclusive pricing on every package we offer. All of our offerings are based on the Standard Package, which includes:

• Antigen design/Bioinformatics
  
• ~15-mer peptide, 10mg at >85%
  
• 5mg uncoupled peptide sent to customer, 4mg coupled to KLH, 1mg for ELISA
  
• Mass Spec and HPLC trace
  
• Three immunizations of two rabbits (New Zealand White - SPF)
  
• 5mL pre-immune serum per rabbit
  
• Two production bleeds (40-50mL antiserum) per rabbit
  
• ELISA on 1st production bleed
  

Many customers also add affinity purification, which tends to remove potential background signal across multiple applications.

Click here for a price list of our available protocols in all species.

Modification Specific Antibodies - Reagents You Can Trust

Post-translational modifications regulate a myriad of biological functions. Studying and tracking these events with antibodies you can trust are a rock you can build the rest of your research on.

We have made thousands of modification specific antibodies, using a myriad of different types of modifications (phosphorylation, methylation, acetylation, cleavage-site, nitrosylation, ubiqination, etc.). The approach that works for all of these is straightforward and essentially the same: Immunize with the target peptide and perform purifications on the resultant sera using columns made with both the target peptide and a negative control peptide or peptides. Different projects can require adjustments to this process, of course, but this general procedure is the start of what we know ensures the most specific resultant end material possible. Why settle for anything less?

Our Modification Specific Protocol Structure

• Antigen design/Bioinformatics
  
• Two peptides at >90%, 14mg modified, 10mg non-modified peptide
  
• 5mg uncoupled to customer, 1mg for ELISA, 4mg column each peptide
  
• 4mg modified-peptide coupled to KLH
  
• Mass Spec and HPLC trace (each peptide)
  
• Three immunizations of two rabbits (New Zealand White - SPF)
  
• 5mL pre-immune serum per rabbit
  
• Two production bleeds (40-50mL antiserum) per rabbit
  
• ELISA on 1st production bleed (each peptide)
  
• Double affinity purification of 40-50mL antiserum to yield modification-specific antibody
  

The Results

An independent study conducted years ago of nine separate peptide providers found the antibody results that derived from using our materials were twice as robust as those of any other vendor. We were also found to have the best customer service of all participating companies.

We relish the opportunity to learn and grow every day by experiencing new things. If we were already the best eight years ago, just imagine how good we are now.

NEP's proprietary peptide array platform offers customers a rapid, cost-effective way to synthesize 96 different peptides. Unique to New England Peptide, Flashpure™ Custom Peptide Arrays are purified using NEP's proprietary reverse phase purification process to remove all non-peptide contaminants. These contaminants can result in false negatives and positives as well as poor peptide solubility. Flashpure™ is included in all arrays at no additional cost. Array peptides are unbound and delivered in 96 individual tubes, in a 96-well plate format. Each plate is individually tested for accuracy and can be used for epitope mapping, peptide libraries, protein characterization and much more. We offer up to 20-mer peptides at 2.5μm scale. We also offer 3 tiers of analysis and delivery is only 2-3 weeks. Using this format, peptides cost as little as $22 each!

• 96 FlashPure™ peptides per plate in 96 individual tubes in 96-well plate format
  
• 1-2mg average for a 15 amino acid peptide (individual weights per tube not provided)
  
• No cross contamination
  
• 2-3 week delivery
  
• QC of each individual plate (analysis tiers listed below)
  
• Biotinylation, Fluorescein, Stable Isotope Labeling and other modifications available
  
• The array is best suited for peptides that are 12-18 amino acids in length. Max length is 24 amino acids
  
• The average purity is of a 15-mer is ~75%. The approximate failure rate per plate is <5%. Failure rate may increase with longer peptides, hydrophobic sequences, and modified sequences
  

Quantitative analysis of peptides is complicated by the presence of water and counterions, which make the net amount of peptide ~60-80% of the measured (gross) weight of a peptide. The water and counterions (usually trifluoroacetate, acetate, or chloride) can be measured separately, but the net peptide content (NPC) can readily be measured by quantitative amino acid analysis (AAA). Stein and Moore developed AAA as analytical technique for aiding protein structure determination, and their incomparable contribution to biochemistry earned them the Nobel Prize in Chemistry in 1972. Stein and Moore’s technique is still the main method used for AAA today and involves the use of a high performance cation exchange separation followed by a post-column derivatization that allows sensitive detection of the amino acids. The average sensitivity using this technique is in the 0.5-1 nanomole range of material.

New England Peptide is a premier provider of AAA services. This gives our customers additional confirmation of amino acid integrity in the final peptide products, as well as quantitative information about the net peptide content of their product. In this way, customers can order an exact net peptide for their sensitive bioassay work. Additionally, the explosion of research in stable isotope dilution (SID)-based mass spectrometry assays for protein quantitation requires AAA to provide exact molar quantitation and/or aliquotting of peptide isotopologues. NEP's NEPTune™ line of peptide products for proteomics applications utilizes AAA to provide accurate and precise AAA measurement of peptide concentrations or amounts for "mass spectrometry assay-ready" custom products.

NEP's amino acid analysis assay is tested daily vs NIST protein standards and maintains a CV of <3%.

New England Peptide, Inc. has a variety of MS technologies in house including MALDI and ES-Ion Trap Mass Spec technologies. Please contact us today for characterization of your peptides or proteins.

• HPLC - determine the purity of your sample
  
• MALDI-MS to provide confirmation of identity and isotopic incorporation to give desired mass shift.
  
• Electrospray Ion Trap LC-MS and MS/MS Fragmentation.
  
• High resolution MS to confirm isotopic incorporation and pattern of peptide.