Frequently asked questions



Lyophilized peptides
For long-term storage of peptides, lyophilization is highly recommended. Lyophilized peptides can be stored for years at temperatures of -20 °C or lower with little or no degradation.

Peptides in solution
Peptides in solution are much less stable. Peptides are susceptible to degradation by bacteria so they should be dissolved in sterile, purified water.

In solution, some slow degradation reactions may take place, the rate of which will be sequence dependent:
- Peptides containing methionine, cysteine, or tryptophan residues can have limited storage time in solution due to oxidation. These peptides should be dissolved in oxygen-free solvents.
- Glutamine and asparagine can deamidate to Glu and Asp, respectively
- Cysteines can undergo oxidative cyclisation to form Cys-Cys
- Charged residues (Asp, Glu, Lys, Arg, His) are hygroscopic (take up water + moisture)

To prevent the damage caused by repeated freezing and thawing of peptides, dissolving the amount needed for the immediate experiment and storage of the remaining peptide in solid form is recommended.

Avoid moisture
As moisture will greatly reduce the long term stability of peptides, peptides should be allowed to equilibrate to room temperature in a dessicator before dispensing, thus avoiding exposure to moisture in the air which will condense on the peptide. Once dispensed, the tube should be gently purged with anhydrous nitrogen or argon, the container recapped, sealed with parafilm and stored at -20 °C.

Peptide solubility characteristics vary strongly from one peptide to another.
Residues such as Ala, Cys, Ile, Leu, Met, Phe, and Val will increase the chance of the peptide having solubility problems.

Solubility properties
The best solvent to use will depend on the solubility properties of the peptide and solvent requirements of your assay. We recommend predicting the physical properties of the peptide, dissolving the peptide as a function of these physical properties and then adapting the solubility results experimentally.

Use distilled solution
In order to reconstitute the peptide, distilled water or a buffer solution should be utilised. Some peptides have low solubility in water and must be dissolved in other solvents such as 10% acetic acid for positively charged peptides or 10 % ammonium bicarbonate solution for negatively charged peptides.

Select the best solvent
Other solvents that can be used for dissolving peptides are acetonitrile, DMSO, DMF, or isopropanol. Use the minimal amount of these non-aqueous solvents and add water or buffer to make up the desired volume.
Always use pure solvent first, then dilute by adding water stepwise until you reach a solvent concentration compatible with your assay.

Use and storage
After peptides are reconstituted, they should be used as soon as possible to avoid degradation in solution.
Unused peptide should be aliquoted into single-use portions, relyophilized if possible, and stored at -20 °C. Repeated thawing and refreezing should be avoided.

Particular cases
For peptides that tend to aggregate (usually peptides containing cysteines), add 6 M urea, 6 M urea with 20 % acetic acid, or 6 M guanidine - HCl to the peptide, then proceed with the necessary dilutions. Please note that urea irreversibly alters the side chain of lysines. If this is to be avoided, use of guanidium chloride is advised.

A major problem associated with the dissolution of a peptide is secondary structure formation. This formation is likely to occur with all but the shortest of peptides and is even more pronounced in peptides containing multiple hydrophobic amino acid residues. Secondary structure formation can be promoted by salts.

The industry standard is to deliver peptides in a lyophilized form and to state the delivery amount as the weight of the lyophilized powder “Gross weight”.

But beside the peptide of interest, the production mix contains other peptidic entities such as truncated peptide forms, deprotected peptides or incomplete peptide sequences. All together these peptidic molecules form the “peptide content”.

The gross weight, in addition to the peptidic weight, contains and is largely influenced by other components such as residual solvent, water and the TFA counter-ion whose molecular mass is high (114Da). Hence TFA which binds to the free N-terminus of the peptide as well as to the basic residues, significantly contributes to the gross weight of the lyophilized material.

Therefore when ordering 1 mg of peptide, you will receive 1 mg of powder which may contain 60-80% peptide.

The net peptide content (NPC) is the fraction of peptidic material present in the lyophilized material.
In combination with the peptide purity, it allows to determine the exact amount of the peptide of interest.

NPC is traditionally measured by amino acid analysis (AAA; limited accuracy but requires a low material amount) or elemental analysis (CHN; requires milligrams of peptide but is more accurate). Both methods measure total peptidic content.

Eurogentec offers Quant-Peptides corresponding to two proprietary peptide quantitation methods (with and without Quant-Tag), offering net peptide content with better accuracy and reproducibility than AAA or CHN.

Thanks to our long experience in anti-peptide antibody programmes, Eurogentec has optimised the use of a combination of specific algorithms for peptide selection.

In our hands, the success rate of immunization using only one peptide per protein and after correct sequence analysis turns around 75 % statistically.

If the antibody production is carried out with two peptides out of the protein sequence, the failure risk will be reduced. The chance of success increases from 75% to about 90-95% (success = the protein is recognised in Western blot).

Request a design

Eurogentec uses solid phase Fmoc chemistry rather than tBOC chemistry. Peptides synthesised by tBOC chemistry require more purification due to the harsh synthesis conditions and specific equipment related to the use of hydrofluoric acid (HF).

All peptides are analysed by MS to confirm the molecular weight. For peptides where a minimum purity has been requested we also run reverse-phase HPLC analyses. The results from these analyses are included on the Technical Datasheet (TDS) supplied with the peptides delivered.

Molarity refers to the molar concentration of a solution, which is the number of moles of solute dissolved in 1 liter of solution, expressed as mol/L, or M.

Molarity [M] = Mass / (Volume x Molar Mass); Mole = Concentration (g/L) x Volume (L)/MW (g/mol)

Given: 1mg of dry peptide powder with MW: 20KDa (Molar mass of peptide is 20 g/mol)
To determine molarity with known mass and known volume
For a 1ml solution of this peptide:
Molarity = Mass (0.001g) / (volume (0.001L) x Molar Mass (MW 20,000) = 50μM

To determine mass to achieve a certain molarity:
If for your assay, you need 0.01mM working peptide solution in 1ml of water, then calculate mass required as follows:

Mass = Molarity x Volume x Molar Mass
Mass = 0.01mM x (1/1000 L) x 20,000g/mol = 0.0002g

Hence, you will need 0.2mg/ml of peptide to have a working solution of 0.01mM.

Peptides are shipped lyophilised at room temperature via express courier. On request, we can also deliver your peptides in solution.

The solubility of a peptide is sequence dependent and often hard to predict; in the most difficult cases, multiple attempts may be necessary to find the best combination of solvents and pH. Please see "How should I solubilise my peptide".

The standard biotin modification does not include a spacer. Spacers are available on request.

There are two types of cyclisations that can be performed:

N-terminal to C-terminal cyclisation
Disulfide bridge cyclisation

N-terminal to C-termal cyclisation is confirmed by a shift in molecular mass of 18 mass units in the Maldi-Tof mass spectrum.

A disulfide bridge cyclisation is confirmed by MS and HPLC before and after the cyclisation step. Although a mass shift of 2 mass units can be difficult to detect for certain peptides, a HPLC shift helps confirming the completion of the reaction.

Yes, we can synthesise wobble peptides. As there are known differences in the incorporation rates of different amino acids, we can compensate for these rates to produce roughly equimolar amounts of each wobble peptide.

We synthesise peptides containing non-radioactive atoms from commercially available building blocks. This includes amino acids containing 13C and/or 15N for NMR or Mass Spectrometry applications for example.

Peptide content is not an indication of peptide purity; these are two different measurements. Purity is determined by HPLC and indicates the presence/absence of contaminating peptides with undesired sequences.

Net peptide content informs on the percentage of total peptide versus total non-peptide components in the lyophilized powder. The net peptide content is traditionally measured by Amino acid analysis (AAA; limited accuracy but requires a low material amount) or elemental analysis (CHN; requires milligrams of peptide but is more accurate). Both methods measure total peptidic content, and none is perfect.

Our peptides are generally purified by preparative reversed phase HPLC, using two tri-fluoro acetic acid (TFA) modified buffers at pH 2. Buffer A is 0.1 % TFA in deionised water and buffer B is 0.1 % TFA in acetonitrile (ACN) at pH 2.

Peptides are dissolved in either straight buffer A, or some amount of buffer B then diluted with buffer A. Sometimes it is necessary to use an organic polar solvent like (formic acid or acetic acid) in DMSO or DMF to aid in the dissolving of hydrophobic peptides but this is done on a case-by-case basis depending on the sequence anaylsis. On rare occasions we can get better solubility and therefore better purification at pH 6.8 so in that case we dissolve the peptide and run the gradient using two alternate buffers.

The pH 6.8 buffers we use are 10 mM ammonium acetate in deionised water (buffer A) or ACN (buffer B). The separation is monitored by UV at 214 nm and fractions are collected and analysed by MADLI-TOF mass spectrometry for product identity and by reversed phase analytical HPLC for purity.

The fractions are then lyophilised to remove the solvents. The fractions that meet the specifications of the order are then combined into one vial. Next, we run a final MALDI-TOF and analytical HPLC on the combined material.

MALDI stands for Matrix Assisted Laser Desorption - Time of Flight. This machine is used to determine the mass of molecules present in a sample.

We can confirm that we have the correct peptide by comparing the theoretical molecular mass of the peptide synthesized with the experimentally determine molecular mass of the synthetic material.

It is very common to see Na (sodium) and K (potassium) adducts in the MALDI spectrum.

The sodium and potassium comes from the water used in the peptide solvents. Even distilled and deionized water has trace amounts of sodium and potassium ions, which can never be entirely removed. These become ionized during the MALDI mass spec process and bind to the free carboxyl groups of the peptide.

Because there is no water purification system that will remove every single sodium or potassium ion from water, seeing the sodium and potassium adducts at times is very common and unavoidable in MALDI mass spec. This is not an indication that the peptide is not pure, nor should it be confused with an incorrect molecular weight.

We usually use peptides that are 13 to 15 amino acids long in length, however shorter and longer peptides have been known to work.

We have successfully raised anti-peptide antibodies to peptides that differ in the presence/absence of a single phosphate (our phospho-specific antibody programme).

Antibodies typically recognise epitopes are typically between 6-8 amino acids, by presenting a peptide that is 15 amino acids long we increase the likelihood of generating useful antibodies but limit the chance of producing a peptide with a secondary structure that might be unrelated to the antigen.

In order to mimic a protein's physical and chemical properties, you should request peptides that have a similar structure and charge to the protein.

For peptide sequences that represent the N-terminus we recommend keeping the N-terminus as NH2 like in the protein, and modifying the C-terminus with an amide group to mimic a peptide linkage.

For peptide sequences that represent the C-terminus we recommend keeping the C-terminus as COOH like in the protein and modifying the N-terminus with an acetyl group to mimic a peptide linkage.

For internal peptides both ends of the peptide should be modified (N-terminal acetyl and C-terminal amide) to mimic both peptide linkages.

The most important factor is that the site of conjugation be not an internal amino acid and that the site of conjugation be specific for a single amino acid.

Internal amino acids and multiple conjugation sites will not present the peptide in a way that is most similar to the natural antigen.

After that the choice of chemistry will be dependent on the type of amino acids present in the sequence.

The international nomenclature used for the sequence termini is the following:

- N-terminus: H means free amine (NH2-), Ac mean acetyl [CH3C(O)-NH-], Pyr means pyroglutamic acid
- C-terminus: OH means free acid (-COOH), NH2 means amide [-CONH2]

- Modifications on the side chain of amino acids are depicted in the parenthesis after the corresponding amino acid. For example; phosphorylated serine = S(PO3H2) or epsilon-N-acetylated lysine = K(Ac)