Peptide Synthesis: Handling and Storage of Synthetic Peptides

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Frequently Asked Questions (FAQ)

References using synthetic peptides and antibodies from LifeTein. Full Publication List of 2017.

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How should I dissolve peptides?

The solubility of a peptide is determined mainly by its polarity. Acidic peptides can be reconstituted in basic buffers, whereas basic peptides can be dissolved in acidic solutions. Hydrophobic peptides and neutral peptides that contain large numbers of hydrophobic or polar uncharged amino acids should be dissolved in small amounts of an organic solvent such as DMSO, DMF, acetic acid, acetonitrile, methanol, propanol, or isopropanol, and then diluted using water. DMSO should not be used with peptides that methionine or free cysteine because it might oxidize the side chain.

Test a portion of the synthesized peptide before dissolving the rest of the sample. Lyophilized peptides should be centrifuged briefly to pellet all the material. You might need to test several different solvents until you find the appropriate one. Sonication can be used to enhance solubility.

  1. First, assign a value of −1 to each acidic residue (Asp [D], Glu [E], and the C-terminal –COOH). Next, assign a value of +1 to each basic residue (Arg [R], Lys [K], His [H], and the N-terminal -NH2), and then calculate the overall charge of the peptide.
  2. If the overall charge of the peptide is positive, the peptide is basic. Try to dissolve the peptide in distilled water if possible. If it fails to dissolve in water, then try to dissolve the peptide in a small amount of 10–25% acetic acid. If this fails, add TFA (10–50 µl) to solubilize the peptide, and then dilute it to your desired concentration.
  3. If the overall charge of the peptide is negative, the peptide is acidic. Acidic peptides might be soluble in PBS (pH 7.4). If this fails, add a small amount of basic solvent such as 0.1 M ammonium bicarbonate to dissolve the peptide, and then add water to the desired concentration. Peptides that contain free cysteines should be dissolved in degassed acidic buffers because thiol moieties will be oxidized rapidly to disulfides at pH >7.
  4. If the overall charge of the peptide is 0, the peptide is neutral. Neutral peptides usually dissolve in organic solvents. First, try to add a small amount of acetonitrile, methanol, or isopropanol. For very hydrophobic peptides, try to dissolve the peptide in a small amount of DMSO, and then dilute the solution with water to the desired concentration. For Cys-containing peptides, use DMF instead of DMSO. For peptides that tend to aggregate, add 6 M guanidine, HCl, or 8 M urea, and then proceed with the necessary dilutions.
  5. Tips: If none of the solvents worked, please try Trifluoroethanol (TFE). Trifluoroethanol may form a solvent matrix for assisted hydrophobic interactions between peptide side chains ( TFE has been shown to induce and stabilize α-helices and to induce β-turns, β-hairpins and also β-strands.TFE disrupts tertiary interactions in proteins by weakening non-polar interactions while preserving secondary structures. TFE is frequently used as a co-solvent in protein folding studies with NMR spectroscopy. A mixture of trifluoroethanol (TFE) or hexafluoroisopropanol (HFIP) and trichloromethane (TCM) or dichloromethane (DCM) was found to be very powerful for dissolving peptides as well. TFE and HFIP have been shown to form clathrate structures starting from 10% HFIP or around 20% TFE.

To prevent or minimize degradation, store the peptide in lyophilized form at −20°C, or preferably −80°C. If the peptide is in solution, freeze-thaw cycles should be avoided by freezing individual aliquots.

  • Positively charged residues: K, R, H, and the N-terminus
  • Negatively charged residues: D, E, and the C-terminus
  • Hydrophobic uncharged residues: F, I, L, M, V, W, and Y
  • Uncharged residues: G, A, S, T, C, N, Q, P, acetyl, and amide
  • RKDEFILGASRHD: (+5) + (-4) = +1 This is a basic peptide. See step #2 above.
  • EKDEFILGASEHR: (+4) + (-5) = -1 This is an acidic peptide. See step #3 above.
  • AKDEFILGASEHR: (+4) + (-4) = 0 This is a neutral peptide. See step #4 above.