The solubility of peptides is sequence dependent. Many peptides will solubilize readily in water or phosphate buffered saline with gentle sonication. If the peptide will not dissolve readily varying the pH can help. If the peptide is acidic, try raising the pH slightly. If it is basic, try lowering the pH. For more hyrophobic sequences, some organic may be necessary to dissolve the peptide. Dimethylformamide(DMF) or Dimethylsulfoxide(DMSO) can be added if it is compatible with your protocols. 1% Sodium dodecyl sulfate, or 8M Urea can also be tried for difficult solubilzations.

Some important things to remember:

1. CNT will contact the customer if there is an extremely hydrophobic sequence that could be problematic during solubilization. Recommended solubilization suggestions will be listed on the Certificate of Analysis, but please don’t hesitate to contact our technical services if questions persist.
2. If it is necessary to add organic to the sequence, add the organic BEFORE adding the aqueous solvent. Organic solvent is generally added to peptides containing hydrophobic amino acids: Val, Ala, Leu, Iln, Phe.
3. A rule of thumb in determining peptide pH is the following: Arg, Lys, His and N-terminal -NH2 all contribute to the basic pH of a peptide. Asp, Glu and C-terminal COOH contribute to the peptide’s acidity. Assigning +1 values to basic residues (including CONH2) and -1 values to acidic residues (including CCOOH) within the peptide will yield an overall acidic/basic rating.
4. If the peptide is basic and does not dissolve in water, try adding 10% acetic acid dropwise with vortexing in between. Only use diluted trifluoroacetic acid when acetic acid has failed, as it can be damaging to cells. For acidic peptides try using 10% ammonium hydroxide.

Many times it is unclear what sequence compositions will be troublesome during synthesis, and while some sequences are relatively easy to synthesize they present unexpected difficulties during purification. However, avoiding certain problematic sequences can significantly reduce peptide cost and shorten delivery time. Some things to try to avoid are:

• Bulky amino acids to the N-terminus of a Proline or multiple Prolines.
• Same consecutive amino acids.
• N-terminal Glutamine and Aspartic acid.
• N-Terminal Methionine or multiple Methionine residues.
• Hydrophobic residues localized together (V, A, L, I, F) and not interspersed with highly charged residues (R, K).
• Proline or hydrophobic residues adjacent to phospho-amino acid.
• Beta-sheet formations as a result of multiple and adjacent residues comprised of Ile, Tyr, Phe, Trp, Leu, Gln, Val and Thr.

Yes. Global Peptide Services offers an aliquot service for $3/aliquot.

>80% Purity

• Peptide blocking studies (non-quantitative)
• Protein electrophoresis applications
• Enzyme-sybstrate studies (non-quantitative)
• Polyclonal Antibody production
• Phosphorylation reactions

>90% Purity

• Quantitative protocols
• Affinity Purification
• In vitro bioassays and studies
• Chromatography standard
• Monoclonal Antibody production
• Enzyme studies

>95% Purity

• NMR studies
• Structural studies

Some deletion sequences that result from incomplete coupling may have similar retention properties to the full-length sequence during RP-HPLC purification. Although the majority of deletion-mers are removed, the remaining few contribute to the overall net peptide impurities. It is important to understand the distinction between net peptide content and total peptide content. Net peptide content is percentage of actual peptide material minus any counter ions, salts, absorbed water or solvents and is usually what is meant when one thinks of “peptide purity.” The total peptide content is the peptide, plus all of the residual components just mentioned that are virtually impossible to completely remove from the dry, purified peptide. It is this material that is referred to in the stated weight of your delivered peptide. Net peptide composes approximately 80% of the total weight and can be accurately determined through Amino Acid Analysis, which is available at cost upon request. To say it another way, RP-HPLC purifies the peptide to yield what is considered the net peptide purity (a percentage ONLY taking into account the amount of full-length vs. non-full-length peptide present). Once the peptide has been lyophilized, the peptide that has a net peptide purity of >95% may reveal through Amino Acid Analysis that the total peptide content is 80%. That means that by weight 80% is pure, full-length peptide and 20% is absorbed solvent and water, counter-ions, and salts. When a customer requests a peptide of >95% purity, only <5% of the peptide is not the full sequence, but the delivered peptide is not 95% peptide by weight. For most applications one doesn’t need to perform Amino Acid Analysis to determine net peptide content for the exact ratio of peptide to non-peptide material for quantification in experimental use.

For maximum stability CNT recommends that peptides be stored lyopholized at -20C. If a peptide is to be stored in solution it is recommended that individual aliquots are frozen to to avoid numerous freeze/thaw cycles that can degrade the peptide.

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 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.

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.
Use the following rules:
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

Synthetic peptides have been successfully produced that exceed 100 amino acid residues. The success of such syntheses however, are entirely sequence dependant and purity would likely be very low. Realistically, going above 40 amino acids in length is often "experimental" territory and will significantly affect cost. GPS regularly produces peptides in excess of 40 amino acids.

CNT uses solid phase chemistry utilizing the Fmoc/tBu protection scheme.

Each custom peptide is supplied with Mass Spectrometry spectrum and Analytical HPLC trace. In addition, a Certificate of Analysis is included which states the essential peptide information and suggested solubility conditions. Each peptide is guaranteed to meet the requirements of the order at the time of delivery or CNT will re-synthesize or re-purify the peptide in question, or refund your money.

Peptides are supplied as a lypholized powder, with a triflouoroacetate counter ion. Other counter ions are available upon request.

LC (long chain) biotin is a biotin molecule spaced away from the peptide with the six carbon spacer -amino-hexanoic acid.

Most of our cyclized peptides are oxidized while the Trp is still protected which insures the Trp remains in its reduced form. The peptide is purified after the cyclization, which eliminates any oxidized Trp. Oxidation of any of the peptide residues can be seen as a mass gain of 16 Amu.

If Cysteines are present store peptides dry. When solubilized use dilute concentration and keep acidified. Addition of a reducing agent such as betamercaptoethanol or dithiothreitol is advisable, if possible.

Cyclization is confirmed by a mass loss of 2 Amu in MALDI-MS and a shift seen in HPLC elution time. Ellman’s test is also used to verify the absence of free thiols when applicable.

CNT recommends using >85% peptide purity for polyclonal antibody production. Although using a lower purity peptide will not affect antibody response, subsequent screening and purification results will be improved with higher purity peptides. For Monoclonal Antibody production, >95% purity is recommended.