Peptide Synthesis

Synthetic peptides

Primm produces and offers custom synthetic peptides with a wide range of scales, purity levels, modifications and labelings, to allow you high performance in your research.Over the last 20 years we have built a solid international reputation as a reference company when it comes to peptide synthesis.

Chemistry

Peptide synthesis can be performed in solution (liquid phase) or in solid phase. At Primm the method of choice is the Solid Phase Peptide Synthesis (SPPS) with the Fmoc-chemistry. The peptide chains are assembled on a solid support starting from the C-terminus and progressing towards the N-terminus, by repeating three basic steps:

  • deprotection of the Fmoc alpha amino group of the amino acid
  • activation of the next protected amino acid, as an active ester
  • coupling

The SPPS method guarantees a cleaner chemistry process because, while the peptide is being synthesized step by step, all soluble reagents can be removed from the peptide-solid support by filtration and washed away. After the desired sequence of amino acids has been obtained, the peptide is removed from the polymeric support via TFA (Trifluoroacetic Acid) cleavage with concomitant removal of the protecting groups on the amino acid side-chains.
To achieve high-performaces, custom peptide synthesis is run on automated synthesizers and Primm is equipped with the most modern and efficient ones.
For very difficult sequences or for special modifiers, sometime the manual synthesis is also applied.

Purity and Scale

Purity

Custom Peptides are purified by Reversed Phase (RP-HPLC). HPLC purification allows the removal of the truncated sequences (deletions), the scavengers and the TFA (Trifluoroacetic Acid) used to deprotect and cleave the peptide from the support (TFA could otherwise be extremely toxic and interfere with the expected activity of the peptide).

Primm offers different purity levels:

  • Crude peptides for preliminary experimental screening (e.g. crude peptide libraries)
  • > 70% for immunological purposes (immunograde peptides)
  • > 95% for biological activity studies (animal studies, cell studies); for biochemical reactions (binding experiments or receptor/ligand studies); for structural studies (NMR, X-ray, MS).An ultra pure level (> 98%) is also available upon request.

Scale

Primm offers a wide range of peptide synthesis scales from 1-2 mg up to hundreds of mg. Larger quantities, yielding up to 1 gram of purified peptides, are also available upon request (bulk synthesis).

Peptide library

Due to the wide-spreading interest in Proteomics, peptide libraries have become an advantageous mean for high throughput screening in studies of protein-protein interaction or peptide/protein cross talking, in antigen and epitope mapping, in protein scanning and characterization and in many other fields.
Primm offers peptide libraries with different features to meet all requirements of your research:

  • Size: 24, 48, 96 or multiple peptides (8-15 aa length)
  • Purity level: crude, >70%, >80% and >95%
  • Format: single tube or 96-well plate
  • Lyophilized or resuspended at the desidered volume/concentration
Primm can help the customer in designing the library that best fit his experiment. Usually peptides are 8-15 amino acids long and supplied in 1-2 mg quantity, but different lengths and quantities are also available upon request.

CRUDE LIBRARY

average purity 60-70%
24-48-96 Crude Peptides, 8-15 aminoacid lenght, 2 mg each, C-terminal amide

  • Crude HPLC & MALDI spec of 5 statistically significant peptides
  • Crude HPLC of 5 statistically significant peptides & MALDI spec of all peptides
  • Crude HPLC & MALDI spec of all peptides
* For non amidated peptides (-COOH free)
24-48-96 peptides library

PURIFIED LIBRARY

average purity >70% >80% >95%

Peptides Library, 8-15 aminoacid lenght, 2 mg each, C-terminal amide*

24-48-96 Peptides, HPLC & MALDI spec of all peptides

*For non amidated peptides (-COOH free)

24-48-96 peptides library

Peptide labeling and modifications

 In addition to our standard peptide synthesis service, Primm offers several peptide modifications for a variety of applications. According to individual needs, we can produce C-amidated, N-acetylated, biotin labeled, fluorescein-labeled, phosphorylated, phosphorylated and cyclic peptides, D-amino acids and MAPs.
Linear peptides can also be bound to a solid support like sepharose for affinity purification of antigen specific polyclonal antibodies (see antibody purification section). Sepharose-peptide affinity columns can also be used to study interactions between immobilized peptide and its ligand or between antisense peptide and the immobilized sense peptide.

Peptide labeling:

Either at the N-term or at the side chain of Lysine residues wherever along the sequence
CHROMOGENIC PROBES:

  • Red region of the spectrum

Lissamine
5(6)-carboxytetramethyl-rhodamine [5(6)-TAMRA]

  • Green region of the spectrum

5(6)-carboxyfluorescein [5(6)-FAM] or 5-carboxyfluorescein [5-FAM]
4-chloro-7-nitrobenzofuran [NBD]

  • Blue region of the spectrum

5-dimethylamino-1-naphtalenesulphonyl [DNS]
FRET PROBES:

  • Fluorophores

MCA, Lys(MCA), Glu(Edans)

  • Quencher

DABCYL, Lys(DABCYL), DNP, Lys(DNP)

Peptide modifications:

  • N-terminal acetylation
    •  N-terminal acetylation

·          C-terminal amidation

  • Linkers: Aminohexanoic Acid/8-Amino-3,6-dioxaoctanoic Acid
  • Phosphorylated amino acids (Phosphopeptides)

·          D-amino acids

  • Methylated peptides
  • Formylation
  • Bromoacetylation
  • Myristoylation
  • Succinylation
  • Cyclic peptides: Lactam (amide bond formation), Disulfide (S-S bond formation)
  • Sepharose-bound peptides
  • Conjugated peptides (to carrier protein OVA, KLH or BSA)

SPECIALS:

  • 4 branched MAP resin
  • Metal chelators for “in vivo” imaging
  • Isotope labeling (stable isotopes for biomolecular NMR)

Conjugation

 Synthetic peptides are normally small molecules, which are not able to elicit a significant immune response. However, when they are coupled to a larger carrier molecule, the immune system will then respond to the hapten-carrier complex producing antibodies against the peptide and the carrier protein. We offer you the choice between the most commonly used carrier-proteins: BSA, KLH, OVA and others upon request.
There are a variety of reagents used for binding the peptide to the carrier:

Glutaraldehyde, which cross-links primary NH2-groups on the peptide to those of the protein.
Sulfo-SMCC (Sulfosuccinimidil 4-N-maleimidomethyl-cyclohexane-1-carboxylate), a heterobifunctional reagent that binds, in a two-step reaction, the primary amines of the carrier protein and the sulphydryl group of the cysteine on the peptide.

For peptide sequences lacking cysteine residues, we suggest the addition of a cysteine either at the N- or C-termini of the peptide depending on several criteria. EDC (1-Ethyl-3-(3 Dimethylaminopropyl) carbodiimide), reacts with a carboxylic group, allowing it to be coupled to the amino group in the reaction mixture

Storage, use and handling

Our peptides are delivered lyophilized in a sealed glass vial and for better stability they should be stored at -20°C. Peptideswhich do not contain cysteine residues, stored lyophilized at -20°C, are stable for years.
Peptides containing sensitive amino acids like cysteine (C), methionine (M), and tryptophane (W) should be stored lyophilized at -20°C under an inert gas in order to avoid oxidation.
Peptides in solution are stable for 1-2- weeks at +4°C, for 3-4 months at -20°C, and for 1 year at -80°C. Repeated freezing and thawing should be avoided. If you have to weigh the peptides, do it quickly and in a glass vial, because they are often very hygroscopic and absorb water rapidly. Before weighing, let the peptide come to room temperature.It is a good idea to test the solubility of your peptide on a small sample and then try to resuspend the peptide in deionized water.

If it is insoluble, add 0,1% NH4OH for peptides with a negative charge and 0,1% TFA for peptides with a positive charge. If the peptide is very hydrophobic, start to dissolve the peptide with small quantities of DMSO and then add the buffer that you would subsequently use in order to reach the desired concentration. In many difficult cases, repeated sonications of 30- 60 seconds might help with solubilization.

 

FAQs

Peptide Service

Q:How can I order?

A: Please, send us your inquiry by form online: New Quotation  to get a quote. Our synthesis specialists will contact you.

Q:Can I order on-line?

A: The creation of Primm On-Line Ordering System (POLOS) for Peptides is in progress; this system will be useful for easy sequences (that is: no longer than 25 amino acids, no special modifications and no special labelings).

Q:How do our prices compare to those of our competitors?

A: Our prices are quite competitive and in accordance with prices from other Peptide Suppliers. It has to be taken into consideration that the price depends very much on the quality of the final product. The critical manufacturing step, to reach a good quality, is the purification, that is the most expensive and time-consuming step. The more a peptide is difficult, the more the purification will be complicated and, as a conseguence, the more the peptide will be expensive.

Q:Is there any limitation on the length of peptides?

A: Sequences less than 4 amino acids can be really problematic during the cleavage and the purification. For this reasons, we prefer to evaluate sequences < 5 amino acids. We don’t make dimers. Orders of peptides between 8 up to 25 amino acids in length are always accepted. Primm will also synthesize sequences up to 40 residues and quotes will reflect the quantity and the purity requirements. For longer sequences, please inquire.

Q:What quantity are we able to produce?

A: Primm can produce peptides from mg to grams and at different purity levels from crude to 95-98% pure.

Q:What if Primm does not succed in making the requested peptide?

A: Primm accepts any order for peptides not longer than 25 amino acid residues. If one sequence cannot be made, Primm will inform the customer in approximately 1 week from the order, suggesting eventual modifications to the sequence. Of course, if the sequence cannot be made, any cost will be charged. For sequences longer than 25 amino acids, Primm staff must see them before accepting the order and the acceptance will be communicated within 24 hours. Again, if Primm won’t be able to make the peptide, the customer won’t be requested to pay any fee. To give you a realistic estimation, the synthesis failure at Primm labs, for peptides shorter than 25 aa, is less than 3%.

Q:What purity levels are available and what’s the best purity level for my application?

A: Primm offers different purity levels starting with > 70% up to > 95%, but it’s also possible an ultra pure level (> 98%) upon request.
Please, refer to the following guideline, to select the most suitable grade of purity for your application:
  • non-sensitive screening assays: crude or purity >70%
  • immunological applications: purity > 70%
  • receptor/ligand studies, bio-assay studies or cell studies (e.g. cellular activation / drug screening in cell culture): purity >95%
  • structural studies (NMR, X-ray, MS): purity >95%

Q:If my peptide is 95% pure, what is in the other 5%?

A: Peptide purity is determined by reverse-phase HPLC. As a limit of peptide synthesis method (= solid phase 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, having similar chromatographic characteristics to the target peptide, remain in the peptide sample and account for the percent of impurities.

Q:How are the peptides supplied and how must be stored?

A: All peptides are supplied lyophilized, unless differently requested. The lyophilized solids should be stored at -20°C (or even better at -80°C) for long time or at +4°C for short time (< 3 months). When solubilized should be stored at -20°C / -80°C for short time (< 3 months).

Q:What Quality Control information is supplied?

A: With the exception of crude peptides , which will only be checked by MALDI-MASS spectrometry, all synthetic purified peptides will be analyzed by HPLC and MS to verify composition and purity. The QC documentation is supplied for every peptide and is enclosed to the peptide-vial.

Q:How long does it take from order to shipment?

A: The manufacturing steps of the peptide-production: synthesis, purification and QC analysis require almost 3 weeks. For very long or hard sequences the production should take up to 4/5 weeks.

Q:What type of chemical modification does Primm provide?

A: We offer mostly of the commercially available modifications such as: Biotin, Fluorescein, Rhodamin, isotopic amino acids, MAP 2 or 4 branched, Phosphorylated residues, Spacers-Linkers, Cys-Cys or head-tail Cycles. We can supply also other modifications upon request.

Q:How do I dissolve the peptide?

A: The solubility information is reported on the data sheet enclosed to the peptide. Please, read this carefully before starting. The solubility greatly depends on various parameters (the primary and second structure, the alternation of neutral, hydrophobic and charged amino acids, the nature of the modifications made, the solvent and so on.). Therefore it’s quite impossible to predict the real solubility of the peptide before its actual production.

Please, consider the following suggestions.
As a general role, always begin by reconstituting a small amount of peptide before committing the entire lot. The most common dissolution process is 1 mg of peptide dissolved in 1 mL of sterile water (avoid reconstituting a peptide in a buffer, such as PBS because salts hinder the solubility). If the small amount of peptide is not perfectly solubilized in that conditions (= just water), try the following steps on the left peptide:
  • Sonication is helpful to dissolve
  • Small amounts of dilute (max 10%) aqueous acetic acid for basic peptides and aqueous ammonia for acidic peptides may help
  • Choose the appropriate solvent. Begin reconstituting at a concentration higher than your desired final working concentration (only after the complete dissolution of the peptide, add water or buffer to reach the desired final concentration; note: salts may cause aggregation!)
  • H2O: for hydrophilic residues (K-R-H-D-E-N)
  • Organic solvents: DMF, DMSO, Acetonitrile or TFA: for hydrophobic residues (A-V-L-I-M-F-W)

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