Frequently Asked Questions
How much do peptides cost?
Synthetic peptides are either procured from commercial sources or made in-house based on the criteria presented earlier (see Technology Section). Commercial peptides are priced according to the actual quote at hand. The quotes from several vendors are solicited for each synthetic peptide request. Selection of the particular vendor is usually based on the optimal combination of pricing, turnaround time, and performance history. The total peptide cost is a sum of vendor charges plus RTB ordering fee of 15% plus optional in-house analysis charges. Alternatively, peptides prepared in-house are priced according to an internally developed pricing schedule.
How long does it take to get my peptide?
Turnaround time for synthetic peptides depends on the sequence length and complexity as well as on purity and amount requested. It varies from 2-3 weeks for routine, short peptides to 5-6 weeks or even longer for more complex sequences or a larger quantity of material. Ordering the peptides-protein conjugates at the same time extends the turnaround time by a week on average.
What should I consider before ordering synthetic peptides for my research?
- Solubility - Would the peptide be soluble enough for your application and/or soluble in HPLC compatible solvents to allow for its purification to the desired degree of purity? Generally, peptides composed of predominantly hydrophobic amino acids ( W,Y, F, L, I, V, M, C, A) tend to have low solubility in aqueous solvents. Hydrophilic amino acids such as R, K, H, S, T, D, or E help in peptide solubility, dependent on their prevalence in the sequence. When in doubt, contact Dr. Lukszo to discuss it further.
- Potential synthetic difficulties - While most peptides can be synthesized and purified successfully, sometimes minor modifications to the sequence may greatly improve the chance of successful synthesis and could shorten the manufacturing process or lower its cost. The typical example is a synthesis of peptides with free carboxylic groups at either Cys or Pro residues at the C-terminus. In both cases successful synthesis requires use of the more expensive 2-chloro trityl resin. However, if the same peptides can be ordered as C-term amides (-Cys-NH2 or -Pro-NH2), then synthesis can be performed on the standard amide resin. Another case of a sequence related problem is a Glutamine at peptide N-terminus. During synthesis and purification N-term Gln undergoes a spontaneous transformation into pyroGlutamic residue, which, depending on the sequence may constitute from several to 50% of total material. While application of special purification conditions may help in separating pyroGlu-peptides from desired Gln-peptides - deleting of that amino acid, or adding an additional amino acid could totally eliminate this problem.
- Labeling of peptides - Attachment of various labels (biotin, fluorescein, etc.) to the peptide is most straightforward when these labels are to be attached at the N-terminus. Attachment at the C-terminus or in the middle of the sequence requires more effort and is also more expensive.
What should I consider before ordering synthetic peptide-carrier protein (KLH, BSA) conjugates?
- Purity of peptides to be conjugated to the carrier protein - While many researchers prefer coupling of the highly purified (>95%) peptides, others use less pure and less expensive materials (>75% or >85%) with reportedly good results.
- Method of conjugation to the carrier protein - A coupling through a SH group of Cysteine residue or through the N-term amino group (with glutaraldehyde) are the most common, with the approximate occurrence ratio of 9:1. In the absence of the naturally occurring Cysteine in the peptide of interest, an extra Cysteine may be added to the sequence at either N-term or C-term to serve as an attachment point. With the glutaraldehyde mediated coupling, the best results are attained in the absence of the internal Lysine in the sequence, as the side amino group of it would also react. Generally, it is believed that conjugation involving Cysteine produces better-defined product.
How should I store my lyophilized peptides when I receive them?
They should be stored in the freezer at -20°C or below immediately after receiving them.
How long can I store peptides in solution?
It is not recommended that peptides be stored in solution. The shelf life of peptides in solution is rather limited, especially for sequences containing Cysteine, Methionine, Tryptophan, Asparagine and Glutamine. Generally, the best approach is to aliquot the necessary amounts of peptide for a few days and relyophilize remaining portions for long-term storage (at -70°C), if necessary.
What is the purity of my peptide and how is it measured?
The purity of a peptide is documented in the QC package included with the peptide and is measured by HPLC. This HPLC purity of peptide reflects a ratio (in %) of area of the correct peak versus the sum of areas for all detected peaks at the given UV wavelength (typically 215nm or 220nm). Thus, contaminants not absorbing at these UV wavelengths will remain undetected. Occasionally, peptide impurities may coelute with the major peak in HPLC obscuring the real peptide purity.
What is a net peptide content?
Most peptide (except those devoid of basic amino acids like Arg, Lys, His or with blocked N-term) exist in form of their salts. Synthetic peptides are most likely purified by HPLC, with trifluoroacetic acid (TFA) as modifier. Therefore, these peptides exist as TFA salts, with each basic amino acid residue (Arg, Lys, His) and N-terminus being protonated, and with trifluoroacetate (CF3COO -) counterions. The net peptide content describes (as a percent or a decimal fraction) how much of the received solid peptide represents pure peptide sequence without conterions. It can be calculated by dividing a molecular weight of peptide (supplied in QC documentation) by a sum of this molecular weight and a number of trifluoroacetate counterions (molecular weight of TFA = 114) attached to it. Thus, for example, a synthetic peptide of molecular weight equal 1445 and containing free N-terminal amino group as well as one Arginine and one Lysine would have theoretical net peptide content equal 1445/(1445 + 3 x 114 ) = 1445/1787 =0.8086 or 80.86%. This means (in theory) that 1mg of the peptide material in a vial actually represents 0.8086mg of the pure peptide. Practically, one would have to take into consideration reported purity of peptide as well as possibility of presence of residual water in lyophilized material. The net peptide content can be most accurately established by a quantitative amino acid analysis. Synthetic peptides, whether from commercial sources or made in-house, are supplied based on a gross peptide weight rather than net peptide content.
How do I solubilize my peptides?
Solubility may vary depending on the nature of the peptide. For valuable guides to peptide solubility, refer to the Resources section.
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