As the amino acid composition determines the properties of every individual peptide, testing of peptide solubility with a small amount of product is recommended!
- Allow the peptide to warm to room temperature (preferably in a desiccator) before reconstituting.
- Always use sterile water or buffer (PBS, Tris or phosphate, pH 7) for preparation of solutions.
- For peptides containing Cys, Met or Trp, that are rapidly susceptible to oxidation, oxygen-free solvents should be used.
- Solubility can also be facilitated by carefully warming (< 40°C) or sonication. If the pH of the solution has to be increased, only very weak bases should be used in order to prevent immediate inactivation by racemisation or side reactions.
Guideline for solubilizing your peptide:
- Peptides that are shorter than 5 residues are generally soluble in aqueous media, except in extreme cases where all the residues are very hydrophobic.
- Hydrophilic peptides containing >25% charged residues and <25% hydrophobic residues also generally dissolve in aqueous media, provided that the charged residues are fairly distributed throughout the sequence. Peptides are generally purified with 0.1% TFA/water and 0.1% TFA/ACN solvent system.
- Hydrophobic peptides containing 50% to 75% hydrophobic residues may be insoluble or only partially soluble in aqueous solutions, even if the sequence contains 25% charged residues. It is best to first dissolve these peptides in a minimal amount of stronger solvents such as DMF, acetonitrile, isopropyl alcohol, ethanol, acetic acid, 4-8M GdnHcl or urea, DMSO (if the sequence does not contain C,W or M), and other similar organic solvents, and then slowly add the solution to a stirred aqueous buffer solution. If the resulting peptide solution begins to show turbidity, you might have reached the solubility limit and it will be futile to proceed. Again, it is important to remember that the initial solvent of choice should be compatible with the experiment.
- Very hydrophobic peptides containing 75% hydrophobic residues will generally not dissolve in aqueous solutions. These peptides generally require initial solubilization in very strong solvents such as TFA and formic acid and may precipitate when added into an aqueous buffered solution. The final peptide solution may require a higher concentration of organic solvent or denaturant, which may not be applicable in biological studies involving live cells.
- Peptide sequences containing a very high (>75%) proportion of S,T,E,D,K,R,H,N,Q or Y are capable of forming extensive intermolecular hydrogen bonding network and have a tendency to form gels in concentrated aqueous solutions. These peptides may have to be treated similarly to step #3.
More detail, see Peptide Solubility