Frequently Asked Questions

vivitide produces and distributes products in two categories: catalog products or custom-produced peptides and antibodies according to client specifications. Each lot is analyzed thoroughly by our QC department and supporting analytical data is reported in the Analytical Data Sheet (ADS) with each shipment. Upon request, we can provide a Safety Data Sheet (SDS) for a given product. Complete physical, chemical, biological, pharmacological, or toxicological properties of the products are not known. Our products are intended to be used by qualified professionals under proper laboratory safety practices in appropriate facilities.

If my peptide is hygroscopic in nature, how should it be handled?

A hygroscopic peptide contains charged amino acids (ex: Arg, Asp, Glu, His, Lys), making it vulnerable to exposure to oxygen which can lead to moisture uptake. To prevent the product from liquefying during the weighing process, it should be allowed to warm to room temperature in a desiccator prior to weighing.

If possible, weigh in a glove box to prevent exposure to oxygen and blanket vial with an inert gas prior to restorage. If this is not an option, weigh product quickly and close lid tightly to reduce exposure to the air. If you do not have ideal weighing conditions for this product, it is best to purchase smaller vial sizes to avoid storage after opening.

Quote and Ordering Information

  • How do I place an order?
  • Do you have distributors?
  • How do I ask for a quote?
  • Where can obtain the vivitide w-9 or find remittance information?

Product Documentation

  • Where can I find a COA, ADS, SDS, or Certificate of Origin?

Custom Peptide Quotes

Storage and Handling

Frequently Asked Questions about Peptides

  • Can you explain the packaging of the Peptide Institute catalog products (bulk, -v, -s products)?
  • AMC or MCA: Which one is it?
  • Where do I find information for an enzyme substrate assay?

Frequently Asked Questions about Solid Phase Peptide Synthesis (SPPS)

Peptide Arrays

Polyclonal Antibody FAQs

Monoclonal Antibody FAQs

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Ordering Information

Q How do I place an order? 

A vivitide currently offers several options for ordering products:

  • Email us at sales@vivitide.com
  • Call us at 1-978-630-0020 (MA) or 1-502-266-8787 (KY)
  • Use our convenient Online Quote System
  • Purchase in our convenient Online Catalog Shop
  • Download an order form and fax it to us at 1-978-630-0021 (MA) or 1-502-267-1329 (KY)
  • Order through a distributor

Q Do you have distributors?

A vivitide can ship and sell directly to your country or, alternatively, a list of distributors can be found here.

Q How do I check the status of my order?

A You may contact our friendly customer service department by email at sales@vivitide.com or by phone (888-343-5974) during normal business hours for information on the status of your work.

Q How do I ask for a quote?

A Custom quote requests can be emailed or requested through a form by using our request form here.

Catalog product quotes can be obtained can be obtained through our inquiry form or from the catalog product page.

Bulk or volume quotes often have increased discounts and can be obtained through our inquiry form or from the catalog product page.

Q Where can I find the vivitide w-9 or remittance information?

A The w-9 is available for download on our Remittance Page, here.

General Questions about Custom Peptides

Q What purity do I need for my experiments?

A 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.

Q What is the maximum peptide length you can produce?

A vivitide routinely synthesizes peptides >70 amino acids in length. The longest peptide (protein) we have made synthetically by solid phase peptide synthesis (SPPS) has been 120 amino acids long. We also offer custom protein synthesis services.

Q What is PepTrend™?

A PepTrend™ is a software program designed in-house 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 us to provide the highest level of service for our customers. As a result, Vivitide can instantly alert customers of potential problems while seeking alternate synthesis strategies well before production starts.

Product Documentation

Q Where can I get my Analytical Data Sheet or Certificate of Analysis?

A Supporting analytical information is reported in the Analytical Data Sheet (ADS) for each product. Catalog product ADS will be eventually availble for download, but are currently included in orders. Due to the sensitive and confidential nature of custom peptides, vivitide does not make this information available online for download as eventually will be with the catalog products.  Instead the ADS is included in each product's packaging.  Should you need another copy, or more information, please contact us via email or phone.

Q What about the Safety Data Sheet?

A The Safety Data Sheet (SDS) for each product may be found on each product's page as a PDF to download.  In the cases where the SDS has not been loaded yet, is not online, or is a custom product, they may be obtained by request via phone or email. Complete physical, chemical, biological, pharmacological, or toxicological properties of the products are not known. The products are intended to be used by qualified professionals under proper laboratory safety practices in appropriate facilities.

Storage and Handling

Peptide Storage and Shipping

Q What are the shipping conditions for the peptide I am receiving?

A Most peptides are shipped at room temperature (RT), and peptides are delivered to you in a lyophilized state. These shipping conditions do not compromise quality since these products are stable at room temperatures and above for as long as several weeks. Once a product arrives at your facility, it should be kept at -20°C for long-term storage.  In a rare instance where a peptide requires shipment at low temperature, a dry ice shipment will be arranged. The cost of the shipment is the responsibility of the customer.  Light-sensitive products will be shipped with a dark bottle.  Oxidation-prone products are handled in an argon gas environment, such as those containing a Met or a free Cys, and we recommend that you do the same.

Q How should I store my peptide?

A We recommend the following:

  • Store in a desiccator or with a desiccant to maintain a dry environment.
  • Store in the freezer at a temperature of -20°C
  • 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°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.

Duration

Storage Recommendation

Long-Term Storage

3 Months - Several Years

Lyophilized powder, frozen and desiccated,

-20°C or cooler

Medium-Term Storage

0-3 Months

Frozen liquid (-20°C) or refrigerated lyophilized powder

Short-Term Storage

Less than 1 Week

Refrigerated liquid or refrigerated lyophilized powder

Q What is the appearance of my peptide?

A The physical state for peptides ranges from amorphous solid to crystalline powder.  Some peptides will appear as a small disk at the bottom of the vial, others may appear fluffy or powdery, and other peptides may not seem visible at all.  Particularly, peptides ordered in small quantities such as 0.1 mg or 0.5 mg, may not be visible, especially through an amber glass vial.

Note: Peptides supplied as white lyophilized (freeze-dried) powder can differ in visual appearance between vialed lots due to various components in the process. For example, during exposure to nitrogen flush, the peptides can settle in different patterns. The freezing cycle can also contribute to visual differences depending on the rate; a slower rate will compact the peptide into a dense form. During the drying process, more water may evaporate, leaving the very little peptide visible to the naked eye. The pressure applied in the process will differ from lot to lot as well. These possible scenarios may change the appearance of the white mass in the vial but does not change the amount of peptide contained in the vial.

Q How stable is the peptide I received?

A In general, peptides in solid form are quite stable.  Synthetic peptides, unlike proteins purified from cells, have an extremely low chance of proteinase contamination.

Peptides are stable for more than one year if they are stored in a lyophilized state at -20° C or below and protected from moisture and light. However, following reconstitution of a peptide in solution, stability and storage time will decrease.

If a peptide has been in solution for an extended period of time, homogeneity of the peptide must be reconfirmed.  Typically, once a peptide is in solution, it should be used within a few weeks, even when stored below -20° C.  For specific information on storage time of a peptide solution, refer to the product sheet that comes with the order.

Conditions that can affect peptide stability include the following:

  • Contamination from microorganisms or metal ions can lead to peptide-bond cleavage. Use sterile buffer or water to reconstitute the peptide.
  • Moisture can lead to hydrolysis of the peptide. The peptide should be allowed to warm gradually to room temperature in a desiccator to reduce condensation of water vapor.
  • Constant freezing and thawing can compromise peptide integrity; therefore, stock solutions should be aliquoted.
  • O2 can negatively affect Trp, Cys, and Met residues in a peptide. If a peptide contains any of these residues, Vivitide will blanket the peptide in argon prior to sealing the vial, or screw-cap bottle.

    Certain amino acid bonds in a peptide are more problematic:

  • Asp-Pro bonds are sensitive to acid cleavage.
  • Asn-Gly and Asp-Gly bonds, and sometimes Asp N-term to short side-chain residues (i.e. Ser, Thr, Ala, Asn), can cyclize to form an aspartimide intermediate which, in turn, can undergo spontaneous changes that can alter the peptide.

Q If my peptide is hygroscopic in nature, how should it be handled?

A A hygroscopic peptide contains charged amino acids (ex: Arg, Asp, Glu, His, Lys), making it vulnerable to exposure to oxygen which can lead to moisture uptake. To prevent the product from liquefying during the weighing process, it should be allowed to warm to room temperature in a desiccator prior to weighing.

If possible, weigh in a glove box to prevent exposure to oxygen and blanket vial with an inert gas prior to restorage. If this is not an option, weigh product quickly and close lid tightly to reduce exposure to the air. If you do not have ideal weighing conditions for this product, it is best to purchase smaller vial sizes to avoid storage after opening.

Q How should I reconstitute a peptide in solution?

A The solubility data for catalog products can be retrieved online. To ensure peptide integrity, these recommendations should be followed:

  • The peptide should be allowed to warm gradually to room temperature in a desiccator in order to minimize condensation of water vapor upon opening the vial or screw-cap bottle.
  • Visually locate the peptide in the container. Tap the vial (vortex or microcentrifuge) to release any product that may have become trapped in the cap.
  • Use sterile buffer or water to reconstitute the peptide. For smaller quantities such as 0.1 or 0.5 mg, it is recommended to use a sterile syringe to inject solvent into the vial as opposed to opening the cap. If DMSO is required for solubilization, be sure to use analytical grade DMSO. DMSO can degrade and become dilute with time because it can take up water from the air so care should be taken when using stock DMSO.
  • Aliquot the remaining peptide solution into single-use sterile glass or high quality polypropylene vials for storage to prevent repeated freezing and thawing which can be detrimental to the integrity of the peptide.
  • Store stock solution in a freezer, at -20°C or below, under dry conditions, at a pH of 5-7.

Q What is peptide purity?

A This is the percentage of peptide that is found in the correct sequence as opposed to truncated, deleted, or incomplete sequences that can arise from peptide synthesis. The purity is determined by high performance liquid chromatography (HPLC).

Q What is the difference between gross peptide weight and net peptide weight?

A Net peptide weight is the weight of the total peptide. Some products from the Peptide Institute are distributed in premeasured vials. These are indicated by the suffix –s or –v in the catalog. The net peptide weight is precisely determined by amino acid analysis after acid hydrolysis, HPLC, and/or UV absorption, and the value is indicated clearly on the vial label. The indicated weight is only for the net peptide molecule, and the weights of any constituents are excluded from the quantity. The amount of usable peptide meets and may exceed its advertised quantity.

Gross peptide weight is the weight of the peptide and peptide impurities as well as non-peptide components.

Peptides manufactured by vivitide are sold in gross peptide weight.

Q Why does my KLH/peptide solution appear cloudy?

A 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.

Q How stable is a peptide?

A Most peptides experience very little degradation over time and are stable for more than one year if they are stored in a lyophilized state at -20°C or below and protected from moisture and light. However, following reconstitution of a peptide in solution, stability and storage time will decrease. We do not test the stability of a peptide solution; therefore, prompt use is recommended. However, if you decide to store a peptide solution for several weeks, you should aliquot out the solution into clean, inert glass or plastic vials to prevent freeze-thaw cycles.

Conditions that can affect peptide stability include the following:

  • Contamination from microorganisms or metal ions can lead to peptide-bond cleavage. Use sterile buffer or water to reconstitute the peptide.
  • Moisture can lead to hydrolysis of the peptide. The peptide should be allowed to warm gradually to room temperature in a desiccator to reduce condensation of water vapor.
  • Constant freezing and thawing can compromise peptide integrity; therefore, stock solutions should be aliquoted.
  • Peptides containing Cys or Met are susceptible to oxidation due to the side chain groups with oxygen. It is advisable to blanket the peptide with argon or nitrogen when the vial is opened. Buffers used to dissolve these peptides should be degassed, either by bubbling argon or nitrogen through the solution for 10 minutes, or by subjecting the solution to high vacuum for 10 minutes using a common ultrafiltration capsule. Peptides containing such amino acids tend to have very short-term stability, and long-term storage is not recommended. In some cases, peptides containing Trp may be hygroscopic and require similar handling methods.

Certain amino acid bonds in a peptide are more problematic:

  • Asp-Pro bonds are sensitive to acid cleavage.
  • Asn-Gly and Asp-Gly bonds, and sometimes Asp N-term to short side residues (Ser, Thr, Ala, Asn), can cyclize to form an aspartimide intermediate which, in turn, can undergo spontaneous changes that can alter the peptide.

Q What do I do with the ends of my peptides - keep them free or block them?

A 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.

You can use the following guidelines:

  • 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

Q What are conditions for shipping for a custom peptide?

A Most custom peptides are shipped at room temperature and delivered in a lyophilized state.  Generally, lyophilized peptides are stable at ambient temperature.  Custom peptides containing Cys or Met are susceptible to oxidation due to the side chain groups with oxygen.  These peptides are packaged under argon gas in order to displace oxygen and reduce likelihood of oxidation.  In some cases, peptides containing Trp may be hygroscopic and require packaging under argon gas as well.

Q What is net peptide content and what does it mean? If my peptide is 95% pure, what is in the other 5%?

A 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 us.

Peptide purity is determined by reverse-phase HPLC using a standard gradient established by vivitide (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.

See also “What is the difference between gross peptide weight and net peptide weight?”

Q How should a peptide be stored?

A Once a product arrives at your facilities, visually inspect it and store at ≤ -20°C.  The peptide should be stored with a desiccant to maintain a dry environment.

Q What salt form should I use for my custom peptide?

A When ordering a custom peptide or a catalog peptide, the salt form of a peptide is important. In simple in vitro experiments, a trifluoroacetate (TFA) salt is usually acceptable. However, when experiments are performed in vivo or in cell culture, one may consider ordering an Acetate (-OAc) or hydrochloride (HCl) counter ion to minimize any issues associated with TFA. There is usually a small charge for exchange of the counter ion, but this is worth it to eliminate some of the associated issues known for TFA especially in cell culture experiments and animal models.

Preparing Custom Peptide Solutions

Q How should a peptide solution be prepared or how do I reconstitute my peptide?

A Since there is not a universal solvent for dissolving every peptide, this crucial step is not always straightforward as it may appear.  Because several solvent systems may be necessary until the desired conditions are achieved, always test a small sample of the peptide to determine the best solvents for complete solubilization.  When testing, begin with solvents that can be easily removed by lyophilization, such as water and acetic acid.  For this reason, it is not recommended to start with buffers which have high salt concentrations.  vivitide also offers solubility testing as an additional service for customers, should you desire it.

  • Bring frozen or refrigerated peptides gradually 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.
  • Choose the appropriate solvent. Begin reconstituting at a higher concentration than your desired final working concentration.

Amino Acid Characteristics

Recommended Solvent

Hydrophilic residues (KRHDEN)

H20

Hydrophobic residues (AVLIMFW)

Low solubility in aqueous solvents; are soluble in organic solvents (DMF, DMSO, TFA, Acetonitrile)

 

A completely solubilized peptide is a clear solution. 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 protonate residues and maximize charge.
  • If negative charges are greater, add dilute base dropwise to deprotonate 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 vivitide Technical Service for assistance, should you have further difficulty.

Determine the overall charge

Charged amino acids aid solubility in aqueous environments.  A sequence with little or no overall charge at any pH is not likely to be water soluble.

    Hydrophobic amino acids:  Ala, Phe, Ile, Leu, Val, Pro, Met, Trp, Tyr, Cys

    Positive Charges:  Lys, Arg, His and Free N-terminus

    Negative Charges: Asp and Glu and Free C-terminus

First, determine if the peptide is charged or neutral by calculating the overall charge of the peptide at pH 7.  Calculate overall charge by using the following values:

    +1 for each basic residue (Lys, Arg, and N-terminus)

    -1 for each acidic residue (Asp, Glu and C-terminus)

    For His, use +1 at pH 6

If the overall charge of the peptides is negative then the peptide is acidic, if it is positive then it is basic, and if zero then it is neutral.

Charged peptides

For acidic peptides (and/or if the total number of charges of the peptide at pH 7 is greater than 25% of the total number of residues): use a small amount of 0.1M ammonium bicarbonate to dissolve the peptide, and then dilute it with water to the desired concentration.  Maintain pH around 7 and adjust pH as needed.  For basic peptides (and/or if the total number of charges of the peptide at pH 7 is between 10-25% of the total number of residues): use a small amount of 25% acetic acid to dissolve the peptide and dilute it with water to the desired concentration.  Adjust pH with 8 M NH4OH to desired pH for oxidation (4-7).  For neutral peptides (and/or if the number of charges is greater than 25% of the total number of residues): use the strategy described for acidic peptides.  Otherwise, the use of organic solvents is recommended.

Hydrophobic or neutral peptides

Hydrophobic peptides containing 50% to 75% hydrophobic residues may be insoluble or only partially soluble in aqueous solutions, even if the sequence contains 25% charged residues.  It is best to first dissolve these peptides in a minimal amount of stronger solvents such as acetonitrile, isopropyl alcohol, ethanol, and/or acetic acid, and then slowly add (drop wise) the solution to a stirred aqueous buffer solution.  If the resulting peptide solution begins to

show turbidity, you might have reached the solubility limit and it will be futile to proceed.  Again, it is important to remember that the initial solvent of choice

should be compatible with the experiment.  It is important to dissolve the peptide completely in the initial solvent (such as acetic acid, acetonitrile) because

the rate of dissolution of peptides into these solvents is usually higher than in a water/solvent mixture.  If a water/solvent mixture is used first to dissolve the peptide, the final volume of solution may be larger than necessary.

Sonication

It may be necessary to sonicate the solution before determining if the solvent choice was appropriate.  Sonication should improve solubilization by breaking the solid peptide into smaller particles.  If the solution gels, becomes cloudy or turbid, or has visible particulates, the peptide has not dissolved completely but is suspended.  At this point, a stronger solvent is necessary and the solvents should be removed.  Begin again with the dry sample.

Liability Disclaimer

All products sold by vivitide are intended solely for laboratory and research use and should not be used in or on human subjects.  User assumes all risk of patent infringement by reason of use of material provided by vivitide. vivitide will not be responsible for damages arising from misuse of any product and is not responsible for the results of research using our products.

General Questions about Catalog Peptides

Q Can you explain the packaging of the Peptide Institute catalog products (bulk, -v, -s products)?

A We carry the full line of quality products of the Peptide Institute of Osaka, Japan.  Those that have a catalog number with a -v and -s suffix are packaged as net peptide and are in injection vials.  The products without a suffix are "bulk" and packaged by gross weight.

Vialed (-v and -s) Products:

A peptide with constant weight is lyophilized and sealed under nitrogen in each vial. The net peptide weight is precisely determined by amino acid analysis after acid hydrolysis, HPLC analysis and/or UV absorption measurement, which is indicated clearly on the label of each vial. The indicated weight is only the net peptide molecule and does not include the weight of any additional constituents (water, acetic acid, and so on). For example, code 4002-v Bradykinin is described as follows:

PBK-4002-v Bradykinin (0.5 mg vial), M.W. 1060.2 g/mol

This indicates that each vial contains approximately 0.5 mg of bradykinin and the exact weight is indicated on the label (for example, 0.53 mg) and the instruction sheet (for example, 0.53 mg, 0.50 μmol). The weight is determined carefully and precisely by experts in our quality control department; therefore, we guarantee the quantity in each vial even if the content seems to be a small quantity.

A peptide solution of a known concentration can be constituted easily by injecting a given volume of a suitable solvent, indicated in the instruction sheet, into the vial using a calibrated syringe and dissolving the contents thoroughly. The peptide content in each vial is relatively small and accurately measured, therefore, the peptide should not be taken out of the vial to prepare a solution with a guaranteed concentration. The instruction sheet can be found on the last page of the ADS/COA.

Bulk Products:

The amorphous powder of each peptide is thoroughly dried over desiccant in vacuo and then weighed into a screw-capped bottle. Thus, the weight indicated on the label represents the gross weight of the amorphous powder, which includes the peptide as well as the accompanying water and acetic acid, if any. This is also called "gross" peptide. The amount of water and acetic acid in each amorphous powder is precisely determined by elemental analysis, Karl Fischer titration or gas chromatography. In some cases, peptides contain hydrochloride, trifluoroacetate, or ammonia instead of acetic acid. The observed value(s) of such accompanying constituent(s) is given in the structural formula of the respective peptide described in this catalog. For example, code 4002 Bradykinin is described as follows:

PBK-4002 Bradykinin (Bulk 100 mg)

M.W. 1060.2 • 120.10 • 54.05 g/mol

The total molecular weight of this amorphous powder is calculated to be 1234.4, which consists of 1060.2 for the net bradykinin molecule, 120.10 for two molecules of acetic acid and 54.05 for three molecules of water. This means that 100 mg of this powder contains net 85.9 mg of bradykinin molecules. Amounts of the accompanying water and acetic acid vary with the lot; the exact value in the purchased peptide is available on request. Given the hygroscopic nature of amorphous powder, precise weighing of a small quantity of peptide is not an easy task.

Q AMC or MCA: Which one is it?

A The fluorogenic compound, 7-Amino-4-MethylCoumarin (CAS Number: 6093-31-2, C10H9NO2), is often called AMC and is used in fluorogenic substrates for the detection of proteolytic enzyme activity. The release of AMC can be detected by fluorescence or absorbance.1 Typically, the free compound is referred to as “AMC”. When it is bound to the C-terminus of an amino acid, it becomes 4-MethylCoumaryl-7-Amide, and vivitide refers to the bound version as MCA, although it is sometimes still identified as “AMC”, even when bound. An example, is the compound, H-Lys-MCA, which is sometimes referred to as H-Lys-AMC.

  1. C.P. Linn et al., Anal Biochem. 200(2), 400 (1992).

Q How do I find Assay Methods for my Enzyme Substrate?

A The substrate assay methods can often be found in published literature or, if available, can be found here:

    Assay Methods for Peptidyl-MCA Substrates

    Assay Methods for MOCAc/Dnp Substrates

    Assay Methods for NMA/Dnp Substrates

    Assay Methods for Peptidyl-pNA Substrates

    FRETS-25Xa

    FRETS-VWF73

or feel free to contact us for more information.

Q What leads to different salt forms of peptides?

A Many catalog and custom peptides are present in the trifluoroacetate (TFA) salt form.  Peptides are often purified by reverse-phase High Performance Liquid Chromatography (rHPLC) using a TFA buffer.  The charge on a free amine in the peptide, either on the N-terminus or in a side chain, attracts the TFA counter ion.  In some cases, TFA salt is not the preference and an alternate salt form is preferred.  An additional step is done by using an ion exchange column to exchange the TFA salt with a preferred salt.  Some examples of alternative salt forms are acetate (-OAc) or hydrochloride (HCl).  Since converting from a TFA salt form to another salt form requires an additional step, there is often an additional small fee.

Frequently Asked Questions about Solid Phase Peptide Synthesis (SPPS)

Q What is a safer alternative to HOBt and 6-Cl-HOBt?

A OxymaPure® is an additive used in carbodiimide-mediated (DIC or EDC-HCl) reactions in the conversion of carboxylic acid to amide, with increased yield and decreased side reactions.   It is a safer and more effective alternative to HOBt and has no transport restrictions. vivitide is an official distributor of OxymaPure®. Read more about OxymaPure® here. Other alternatives to HOBt and 6-Cl-HOBt are HCTU or HATU. View all peptide synthesis reagents here.

Q Where can I find more information on CLEARTM Resins?

A CLEAR (Cross-Linked Ethoxylate Acry- late Resins) was developed by George Barany and Maria Kempe at the University of Minnesota. These products retain the highly desirable solvation properties of polyethylene glycol (PEG) or of PEG-linked products but with greater convenience. Unlike resins for conventional liquid phase synthesis, CLEAR resin is a highly cross-linked solid support. It is produced in a bead form using a large-scale polymerization process developed at vivitide. Read more about CLEAR resins here.

Q Do you carry a polymer-supported oxidant to help prepare disulfide bonds?

A CLEAR-OXTM, is a polymer-supported oxidant, which combines the power of solid phase chemistry with the versatility of solution-phase reactions. CLEAR-OXTM is a highly effective, polymer-supported reagent for the formation of disulfide-bonds. CLEAR resin is the polymer of choice due to its compatibility with both aqueous and organic environments. A lysine-preformed cyclic Ellman’s reagent [5,5’-dithiobis(2-nitrobenzoic acid) = DTNB] is covalently attached to a CLEAR polymeric support with a β-alanine spacer to yield CLEAR-OXTM. Since the mechanism is based on peptide capture, sensitive residues such as Tyr, Trp, and Met are not affected, leading to increased purity and yield. These improved synthetic conditions allow for facile removal of the oxidant. Read more about CLEAR-OXTM and download resin conditioning and general methods here.

Q How do I choose an appropriate substitution for my resin of interest?

A Mesh is the number of square openings in a linear inch of screen or sieve. As the mesh number increases, the number of openings also increases while the size of the openings decreases. Therefore, larger mesh numbers correspond to smaller bead or particle sizes for a resin.

US Mesh

Inches

Microns

Millimeters

100

0.0059

149

0.149

120

0.0049

125

0.125

140

0.0041

105

0.105

170

0.0035

88

0.088

200

0.0029

74

0.074

230

0.0024

63

0.063

270

0.0021

53

0.053

325

0.0017

44

0.044

400

0.0015

37

0.037

 

Q What does the mesh number mean for a resin?

A The higher the substitution, the greater the points of attachment points there are. However, higher substitutions can lead to decreased synthesis efficiency and cause aggregation with peptides containing long or difficult sequences. This is caused by increased steric hindrance that accompanies high density of peptide chains present in the matrix. Lower substitutions are also better to use for cyclizing linear peptides.

Q The substitution for resins is shown in the units, meq/g. How do you convert this to mmol/g an appropriate substitution for my resin of interest?

A No conversion is required since meq/g is equivalent to mmol/g.

Peptide Arrays

Q How can New England Peptide help me with my HTS?

A 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.

Q Which array analysis tier is right for me?

A

  • 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.

Polyclonal Antibody FAQs

Q Which antibody package is right for me?

A 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.

Q 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.

A What species should I choose for my antibody production?

Q 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.

Q Why do I need two rabbits?

A 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.

Q Why should I use a peptide for antibody production?

A 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.

Q How do you choose the best antigen?

A 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.

Q Can I inject more than one peptide into the same set of animals?

A 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.

Q What chemistry do you typically use for carrier protein conjugation?

A 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 sulfhydryl 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.

Q What do I do with my pre-immune serum?

A Pre-immune serum is a great negative control for a wide variety of experiments.

Q How do you test for reactivity?

A 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.

Q How much antibody should I expect to receive?

A 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 vivitide Standard Antibody Package provides ~80mL antisera. The vivitide Affinity Purified Antibody Package often yields >10mgs of purified antibody in addition to 40mL antisera.

Q How do I store my antisera / antibody?

A 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.

Q How long can I expect my rabbits to produce good antibody?

A 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.

Q Will I be able to use my antibody in multiple applications?

A We design the peptide so that it gives our antibodies a very good chance 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.

Q What are your accreditations?

A 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.

Monoclonal Antibody FAQs

Q What should I use as an antigen?

A We can either design and manufacture a specific peptide antigen or use another type of antigen (protein, viral, etc.) that you provide. We will work with you to determine the best antigen for your project needs.

Q If I send you material, how much do you need and how should I send it?

A 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).

Q What strain(s) of mice should I use?

A 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.

Q What screening method does vivitide use?

A In the standard monoclonal package, vivitide 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.

Q What will I receive at the end of the package?

A 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.

Q What is vivitide's policy of ownership and confidentiality?

A The customer owns all antibody material produced under contract to vivitide. Each order is handled with strict and complete confidentiality. We can also work together to execute a Confidential Disclosure Agreement (CDA).