Peptide solubility guidelines
Smart Bioscience 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 calculations, which can introduce experimental error into data or result in experimental failure. Finding the ideal solvent in which to dissolve your peptide is often a challenge. Even if the solubility of Smart Bioscience peptides is tested, the guidelines below may be helpful in preparing peptide solutions.
General solubilization procedure
- Work on a small portion to test the solubility of the sample before dissolving the entire sample (take it into account when defining the quantity and aliquots to order. We recommend to keep 1 tube of 1 mg dedicated to this)
- Centrifuge all product preparations before use (10000 x g 5 min) to allow the whole powder going down.
- Allow the peptide to warm to room temperature before reconstituting.
- Try to use sterile and ideally oxygen-free water or buffer (Tris or phosphate, buffer at pH 7) when preparing solutions.
- Sonication notably improves the solubility of peptides (10sec x 3, ice between). Even hard to solubilize peptides can be soluble in water with sonication.
- Gentle warming can help
- A peptide properly solubilized will give a transparent solution. On the contrary, the solution will be cloudy or you can even saw some particulates.
|A well solubilized peptide looks transparent, on the contrary it looks cloudy|
Peptide properties calculation & associated solubility
Before solubilizing any lyophilized peptides, their amino acid composition can help you in predicting the solubility.
First you have to determine if the peptide is acid, basic or neutral:
- 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<6 and zero at pH >6.
- Count the total number of charges of the peptide at pH 7 (all D, E, K, R, C-terminal COOH, and C-terminal NH2).
- Calculate the overall net charge of the peptide.
Charges < 0: Acid peptide.Try to solubilize it with a basic solution by adding 10% NH4OH or ammonium bicarbonate in your buffer.
Charges > 0 : Basic peptide. Try to solubilize it with an acid solution by adding 10% acetic acid, 0.1% TFA, 0,1% formic acid in your buffer.
Neutral peptide: if the peptide contains > 25% charged residues (e.g., D, K, R, H and E) it is generally soluble in water or aqueous buffers. Below 25% charged residue, it is recommended to use organic solvents (DMSO, ACN, DMF).
Oxidation sensitive peptides
Cys, Met or Trp are susceptible to oxidize rapidly. Use oxygen-free solvents and avoid the use of DMSO when handling these peptides. Cys containing peptides can also form unexpected disulfide bridges (intra or inter chain) that can be prevented by working in an acidic solution using DTT (dithiothreitol)
Below five residues, peptides 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, organic solvents will probably be necessary.
Highly hydrophobic peptides
Peptides containing more than 50% hydrophobic residues (W, L, I, F, M, V, Y, P, A) are generally poorly soluble in aqueous solutions. We recommend to dissolve the peptide in 100% ogranic solvent (DMSO, DMF or acetonitrile). Once it is solubilized, add water slowly or buffer up to the desired concentration is reached. If the peptide precipitates, we recommend to lyophilizate the peptide and start again with a lower dilution. DMSO is the preferred organic solvent for working in cellular biology because of its low toxicity. If DMSO interferes with your experimental system, use DMF or acetonitrile as an alternative.
Addition of chaotropic compounds such as 6 M urea, 6 M urea with 20% acetic acid, or 6 M guanidine can facilitate in breaking up hydrophobic interactions or reduce the aggregation of peptides by disrupting hydrogen bonding network.