Peptide solubility guidelines

IC-PEPTIDE's custom synthesized peptides are delivered as lyophilized powder in 2 mL microtubes, that needs to be reconstituted. The solubilization is a critical step for a successful peptide assay. Improper peptide solubilization results in inaccurate peptide concentration, which can introduce experimental errors into data or lead to experimental failure. Finding the ideal solvent to dissolve your peptide is often a challenge. Even if the solubility of IC-PEPTIDE peptides is tested, the guidelines below may be helpful in preparing peptide solutions.

General solubilization procedure

Ask now for your peptide solubility calculator by submitting a request through the form on the right side of the page. You will receive it by email. A peptide properly solubilized will result in a transparent, particle-free solution.

Test peptide solubility using a small amount of the product, rather than the entire sample.
Centrifuge to spin down any powder on the tube sidewall before use (10000×g for 5 min).
Allow peptides to warm to room temperature before reconstituting.
Use sterile and ideally oxygen-free water or buffer (Tris or phosphate, buffer at pH 7) when preparing peptide solutions.
Briefly sonicate the mixture to aid peptide dissolution (3× 10 sec, ice between).
If precipitation occurs, a gentle warming of the peptide solution would be helpful.

Peptide properties calculation & associated solubility

The amino acid composition can help predict the solubility of a peptide. Therefore, before choosing a solvent and dissolving the peptide, the sequence should be studied to determine whether the peptide is acidic, basic or neutral.

Charges less than 0: Acidic peptide; try to dissolve it in a basic solution by adding 10% NH4OH or ammonium bicarbonate in your buffer.
Charges greater than 0: Basic peptide; try to dissolve it in an acidic solution by adding 10% acetic acid, 0.1% TFA, 0.1% formic acid in your buffer.
Neutral peptide: if the peptide contains greater than 25% charged residues (e.g., D, K, R, H and E), it is generally soluble in water or aqueous buffers. Below 25% charged residues, it is recommended to use organic solvents (DMSO, ACN, DMF).

Calculation steps:

Assign a value of -1 to each acidic residue (D, E, and C-terminal COOH).
Assign a value of +1 to each basic residue (K, R and the N-terminal NH2).
Assign a value of +1 to each H residue at pH less than 6 and zero at pH greater than 6.
Count the total number of charges of the peptide at pH 7 (D, E, K, R, C-terminal COOH, and N-terminal NH2).
Calculate the overall net charge of the peptide.

Oxidation sensitive peptides

Cys, Met or Trp-containing peptides are susceptible to oxidation. Therefore, it is recommended to dissolve them in oxygen-free buffers. Moreover, DMSO should be avoided when handling oxidation-sensitive peptides. Cys-containing peptides can form unexpected disulfide bonds (intra or interchain) that can be prevented by using reducing agents, such as DTT (dithiothreitol).

Short peptides

Very short peptides consisting of less than five residues are usually soluble in water or aqueous buffer, except when the entire sequence consists of hydrophobic amino acids (e.g., W, L, I, F, M, V, Y). In this case, the use of organic solvents is necessary.

Highly hydrophobic peptides

Peptides containing 50% or more hydrophobic residues (W, L, I, F, M, V, Y, P, A) are generally poorly soluble in aqueous solutions. We recommend to dissolve hydrophobic peptides in 100% organic solvent (DMSO, DMF or acetonitrile) and subsequently dilute with water or buffer to the desired concentration. If peptides precipitate, they need to undergo a freeze-drying step before attempting another solubilization to reach a lower dilution. DMSO is the ideal organic solvent for biological applications because of its low toxicity. If DMSO interferes with your experimental system, use DMF or acetonitrile as an alternative. Addition of denaturing agents, such as 6M urea, 6M urea with 20% acetic acid, or 6M guanidine, can help in reducing the aggregation of peptides by disrupting hydrogen bonding network. However, these compounds can interfere with most biological systems and therefore their application is rather limited.

IC-PEPTIDE development services

To go further, IC-PEPTIDE proposes a peptide solubility study service. We can also study the stability of your peptide in different conditions.