Custom Peptides

General Questions about Custom Peptides

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.

What is the maximum peptide length you can produce?

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.

What is PepTrend™?

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

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

Supporting analytical information is reported in the Analytical Data Sheet (ADS) for each product. Due to the sensitive and confidential nature of custom peptides, vivitide does not make this information available online for download as 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.

What about the Safety Data Sheet (SDS)?

The SDS 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

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

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.

How should I store my peptide?

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

What is the appearance of my peptide?

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.

How stable is the peptide I received?

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.

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.

How should I reconstitute a peptide in solution?

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.

What is peptide purity?

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).

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

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, unless requested as net peptide.

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.

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.

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.

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

What are conditions for shipping for a custom peptide?

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.

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

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?”

How should a peptide be stored?

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.

What salt form should I use for my custom peptide?

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

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

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.

Peptide Arrays

How can vivitide help me with my HTS?

In order to satisfy the growing requirements of the proteomics boom, vivitide 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.