Dissolution of polypeptides
Each amino acid has inherent chemical properties and the solubility of a peptide depends on its sequence and modifications. We recommend taking a small amount of the peptide for solubility testing before dissolving all the peptides.
Dissolving peptides is a very important step. Improper solubilization may result in peptide loss or experimental failure. The solubility of peptides mainly depends on the sequence of the peptide, so pay attention when designing the peptide. It is recommended that the peptide sequence should contain 20% charged residues to increase its solubility.
Before use, customers can follow the following three basic principles to choose a suitable solvent to dissolve the peptide.
First, the chosen solvent must be able to fully dissolve the peptide; Second, the chosen solvent must be compatible with the experimental conditions; Finally, the chosen solvent must not react with or degrade the peptide.
As long as the amount of peptide allows, you can first dissolve a small portion of the peptide and then dissolve the entire sample. If you need to recover the peptide from a solvent, you can choose an initial solvent that makes it easy to remove after lyophilization.
The following suggestions may help you dissolve peptides:
Peptides with less than 5 amino acids are generally soluble in aqueous solutions, and strongly hydrophobic amino acids (W, I, L, F, M, V, Y) except.
If the charged amino acids are evenly distributed throughout the sequence, then contains >25%Charged amino acids (E, D, K, R, H) of hydrophilic peptides and containing <25%Peptides with hydrophobic amino acids are generally soluble in aqueous solutions. The purification system for peptides is generally 0.1% TFA/Water and 0.1% TFA/ ACN. Therefore, if the peptide is dissolved in a buffer with no or very weak buffering capacity, the result may be an acidic peptide solution. When using other methods to dissolve peptides, be sure to use a solvent that is pH The value is nearly neutral. acidic peptide (E+D The number of residues is more than K+R+H number of residues) and basic peptides (K+R+H The number of residues is more than E+D number of residues) in neutral pH value conditions than in acidic pH value is easier to dissolve.
For hydrophobic amino acid content is 50% ～75%peptide, even if the sequence contains >25%Charged amino acids may also be insoluble or partially soluble in water. It is best to first dissolve the peptide in a minimum amount of a strong organic solvent such as DMF, ACN, Isopropyl alcohol, ethanol, acetic acid, 4-8 M GdnHCl, urea, or DMSO (not in sequence C, W, M ), and other similar organic solvents. It is best to slowly add the organic solvent that dissolves the peptide into the aqueous solution drop by drop. If the solution becomes turbid, it may have reached the solubility limit and there is no use in continuing to dissolve it. It should be noted that the solvent initially selected should be compatible with the experimental system.
Hydrophobic amino acid content >75%Strongly hydrophobic peptides are generally also insoluble in aqueous solutions. Such peptides should also be treated first with strong organic solvents (e.g. TFA, Formic acid) dissolves and may also precipitate if added to an aqueous solution. The final peptide solution may require high concentrations of organic solvents to denature it, and organic solvents are generally not used for biological function studies in living cells.
S, T, E, D, K, R, H, N, Q, Y High content (>75%) Polypeptide sequences tend to form redundant intermolecular hydrogen bond networks and easily form gels in concentrated aqueous solutions. Such peptides can be solubilized as described in step 3. In order to reduce possible problems during dissolution, it is recommended that customers pay more attention to the design and sequence to improve the solubility of the peptide.