How are the peptides synthesized?
Fmoc chemistry is used to synthesize the peptides on a solid support from the C-terminus to the N-terminus of the sequence.
What are the right C- and N-terminal endings of the peptides?
This depends on the application you have in mind for your peptides. It may be a good idea to choose the terminal ends of the peptide dependent on the natural occurrence of the sequence:
1. The peptide(s) should mimic an internal sequence of a protein.
The peptide(s) should not be charged at the ends. The N-terminus of the peptide(s) should be acetylated and the C-terminus should be an amide.
2. The peptide sequence is the C-terminal end of a protein.
The C-terminus should be the free acid and the N-terminus should be acetylated.
3. The peptide sequence is the N-terminal end of a protein.
The C-terminus should be an amide and the N-terminus should be in the natural free amine form.
• For cytotoxic T-cell epitope studies the peptides should have both, a free amino group at the N-terminus and a free acid at the C-terminus. These ends are the natural equivalents to the peptide fragments, processed intracellularly from whole proteins.
• Peptides with acetylated N-terminus and an amide as C-terminus are more resistant to exopeptidases, which may be an important factor regarding the time a peptide will be functional in a biological assay.
• Biotinylated peptides can be useful in combination with streptavidin coated surfaces (e.g. beads, plates or microarrays).
• Peptides with a thiole group (e.g. cysteine) can be directly bound to gold surfaces or to amino groups by the use of bifunctional crosslinkers.
How can I estimate the solubility of a peptide?
It is often difficult to predict the solubility of a peptide sequence. Some information about choosing a good peptide sequence can be found here: Peptide Sequence Design.
How are the peptides supplied?
The peptides are delivered lyophilized. Ambient temperature should not be a problem for the lyophilized peptides. However, for maximum stability the peptides should be stored at -20°C.
How do I store my peptides?
The lyophilized peptides should be kept in a cool, dark place. Long term storage of the peptides should be done in a freezer at -20 to -80°C. Most peptides stored in this way will remain stable for several years.
Peptides in solution are not as stable as in the lyophilized form. Peptide solutions should be neutral to slightly acidic (pH 5-7) and stored frozen at -20°C. To avoid repeated freeze-thaw cycles it is recommended to divide the stock solution into aliquots.
• maintain sterile conditions
• Cys, Met and Trp residues tend to oxidize (oxidation-rate increases with pH)
How do I solubilize my peptide(s)?
The solubility of peptides is strongly dependent upon the peptide sequence. For some very hydrophobic sequences it may not be possible to use aqueous solutions without any additional organic solvents.
1. Try to dissolve the peptides in sterile water with sonication (1-10 mg peptide/ml).
2. If this fails, add acetic acid up to a total concentration of 10% (v/v) for basic peptides or aqueous ammonia for acidic peptides and sonicate. (Count the number of basic (R, H, K and free N-terminus) and acidic residues (D, E and free C-terminus) of the peptide)
Examples:
Ac-HN-RFREQIVKPFK-CONH2 -> 0-1+0-1+1+0+0+0-1+0+0-1+0 = -3 -> basic peptide
H2N-FVQADIDYIT-COOH -> -1+0+0+0+0+1+0+1+0+0+0+1 = +2 -> acidic peptide
3. For peptides that remained insoluble add organic solvents such as acetonitrile, DMSO or DMF up to a concentration of 20% (v/v).
NOTE: The use of organics such as acetonitrile, DMF, DMSO etc. may interfere with some biological assays. If DMSO is used, peptides with Cys, Met and Trp oxidize faster!
My peptide has a purity of 95% - what are the other 5%?
The peptide purity gives the percentage of the correct sequence in a given sample. The rest of the sample consists of truncated or deletion sequences.
What is the net peptide content of my peptide(s)?
The weight of the lyophilized peptide composes of the peptide and water weight. The water and counter-ion content vary widely, depending on the given peptide sequence. Typically 90% are peptide and the other 10% are water and counter-ions. For some highly charged peptides the water and counter-ion content can increase up to 30%.
What is the length of the peptides you can synthesize?
The minimum length of peptides should not be less than 5 amino acids. For shorter peptide sequences cleavage from the synthesis resin and following purification can cause problems.
The standard maximum lengths are peptides with up to 40 residues. However, upon request we will synthesize longer peptides (>60 amino acids).
