Peptide Solubility Based on experience, dissolving peptides is an important part. Improper dissolution can result in peptide loss or experimental failure. The solubility of the peptide depends primarily on the sequence of the peptide, so if the experiment allows, at least 20% of the charged residues in the peptide sequence should be included to increase its solubility.
Prior to use, customers can follow three basic principles to select the right solvent to dissolve the peptide. First, the selected solvent must be sufficient to dissolve the polypeptide. Secondly, the selected solvent can be compatible with the experimental conditions. After the Zui, the selected solvent cannot react with the peptide, nor can the peptide be degraded. As long as the amount of peptide allows, it can be dissolved with a small amount of peptide, and then all samples are dissolved. If it is desired to recover the peptide from the solvent, an initial solvent can be chosen to facilitate removal after lyophilization.
The following suggestions may help you dissolve peptides:
1. Peptides with less than 5 amino acids are generally soluble in aqueous solutions, with the exception of strongly hydrophobic amino acids (W, I, L, F, M, V, Y).
2. If charged amino acids are evenly distributed throughout the sequence, hydrophilic peptides containing >25% charged amino acids (E, D, K, R, H) and peptides containing <25% hydrophobic amino acids are generally soluble in aqueous solutions. The purification system for the polypeptide is typically 0.1% TFA/water and 0.1% TFA/CAN. Therefore, if the peptide is dissolved in a buffer that has no buffering capacity or a weak buffering capacity, the result may be that the peptide solution is acidic. When using other methods to dissolve the peptide, make sure that the pH of the solvent is near neutral. Acidic peptides (more E+D residues than K+R+H residues) and basic peptides (K+R+H residues more than E+D residues) at neutral pH It is more soluble at acidic pH.
3. Peptides with a hydrophobic amino acid content of 50% to 75% may be insoluble or partially soluble even if the sequence contains 25% charged amino acids. Zui is good to dissolve the peptide in a small amount of strong solvent (such as: DMF, ACN, isopropanol, ethanol, acetic acid, 4-8M GdnHCl or urea, DMSO (without C, W, M in the sequence), and other similar The organic solvent, zui is good to slowly add the organic solvent that dissolves the peptide to the aqueous solution. If the solution becomes cloudy, it may reach the solubility limit, and it is useless to continue to dissolve. It should be noted that the solvent that Zui chooses should start. Compatible with experimental systems.
4. Strong hydrophobic peptides with a hydrophobic amino acid content of >75% are generally not soluble in aqueous solutions. Such peptides should also be first dissolved with a strong solvent (eg, TFA, formic acid), and precipitation may occur if added to an aqueous solution. The peptide solution after zui may require a high concentration of organic solvent to denature it, and the organic solvent is generally not used for the biological function study of living cells.
5. S, T, E, D, K, R, H, N, Q, Y content (>75%) of the polypeptide sequence easily form redundant intermolecular hydrogen bond network, easy to form a gel in concentrated aqueous solution . Such peptides can be solubilized by the method described in Step 3.
In order to reduce the problems that may occur in the dissolution, it is recommended to consider both the design and the sequence to improve the solubility of the peptide.
Peptide Dissolution Methods The solvent used to solubilize synthetic peptides has been a problem for peptide workers. Depending on the amino acid sequence, the following methods can be used to dissolve the synthetic peptide. Zui is good to dissolve a small amount of peptide with an aqueous solution first, do not dissolve all the samples at once.
1. Set the values ​​of the acidic amino acids Asp (D), Glu (E) and C-terminal -COOH. to -1.
2. The value of the basic amino acids Arg (R), Lys (K), and His (H) is set to +1.
3. Calculate the total amount of charge in the peptide.
4. If the total charge of the peptide is positive, the peptide is basic and can be dissolved in water first. If the peptide is insoluble in water, it can be dissolved with 10% acetic acid or a higher concentration of acetic acid. If the peptide is not soluble at all, add TFA (<50 ul) to help dissolve and dilute to 1 ml with deionized water.
5. If the total charge of the peptide is negative, the peptide is acidic and can be dissolved in water first. If the peptide is insoluble in water, add NH4OH (<50 ul) to help dissolve and dilute to 1 ml with deionized water.
6. If the total charge of the peptide is zero, the peptide is neutral. The neutral peptide needs to be dissolved by an organic solvent such as acetonitrile, methanol or isopropanol, or a denaturing agent (for example, urea, guanidine hydrochloride, etc.) may be added.
Example:
KRLMKSIEVIMPL: (+3) + (-2) = +1. This peptide is alkaline. See step 4 above.
LVKMKSIEDPDCE: (+2) + (-5) = -3. This peptide is acidic. See step 5 above.
MVSRKDLVEHRDM: (+4) + (-4) = 0. This peptide is neutral. See step 6 above.
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