The solubility of polypeptides depends largely on the polarity of the polypeptide. Acidic proteins dissolve in alkaline solutions, while alkaline proteins can dissolve in acidic solutions. Hydrophobic polypeptides and neutral polypeptides containing a large number of uncharged polar amino acid residues or hydrophobic amino acids can be dissolved in a small amount of an organic solvent, such as DMSO, DMF, acetic acid, acetonitrile, methanol, propanol, or isopropanol, and then diluted with water (distilled water). Peptides containing methionine or cysteine should not be solubilized in DMSO because DMSO may cause oxidation of the side chains.

Peptide solubilization test: A small portion of the peptide is taken for peptide solubilization test before the peptide is solubilized. You will need to test several different solvents until you find the most appropriate one (based on experimental needs and solubility tests). Sonication helps to break up the particles and increase solubility. (Note: sonication can cause solution heating and peptide degradation.)

1. Assign a value of -1 to each acidic amino acid, including aspartic acid (D), glutamic acid (E), and the C-terminal -COOH. assign a value of +1 to each basic amino acid, including arginine (R), lysine (K), histidine (H), and the N-terminal -NH2. and then calculate the charge of the entire peptide.

2. If the charge of the entire peptide is positively charged, the peptide is basic. Try to dissolve the peptide in distilled water first; if it is insoluble in water, then try to dissolve it in a small amount of 10%-25% acetic acid. If it still fails, add some TFA (10-50 microliters) to increase the solubility, and then dilute it with water to the desired concentration.

3. If the entire peptide is negatively charged, the peptide is acidic. Try to dissolve it in distilled water first; if it is insoluble in water, try to dissolve it in PBS (PH 7.4), and if it is insoluble again, add a small amount of an alkaline solvent, e.g., 0.1 M ammonium bicarbonate, and then dilute with water to the desired concentration. Peptides containing free cysteine should be dissolved in degassed acidic buffer, because sulfhydryl groups are rapidly oxidized to disulfides when the pH is greater than 7.

4. If the charge of the entire peptide is zero, the peptide is neutral. Neutral peptides are usually soluble in organic solvents. First, try adding a small amount of acetonitrile, methanol, or isopropanol. For highly hydrophobic peptides, a small amount of dimethyl sulfoxide can be used to dissolve and then diluted with water to the desired concentration. For peptides containing free cysteine, DMF rather than DMSO is required.For peptides with a tendency to aggregate, add 6 M guanidine hydrochloride or 8 M urea, then dilute as necessary.

To prevent or minimize peptide degradation, store peptides as a lyophilized powder at -20°C, -80°C is preferable. If solution peptides need to be stored, it is best to store them in small samples to avoid repeated freezing and thawing. One sample thawed and unused should be thrown away. Bacterial degradation can sometimes be a problem with solution peptides, so please dissolve the peptide in sterile water or filter the peptide solution to remove bacteria.

Basic amino acids: K, R, H, N-terminus

Acidic amino acids: D, E, C-terminus

Polar neutral amino acids: S,N,Q,T,C,P

Nonpolar hydrophobic amino acids: G, A,M,F,W,V, Y,I,L Acetyl, amide groups

Examples:

KKEEFILGASRHD: (+5) + (-4) = +1 Considered basic peptide, see step 2

ERDDFILGASEHK: (+4) + (-5) = -1 Considered acidic peptide, see step 3

AKDEFILGASEHR: (+4) + (-4) = 0 is considered a neutral peptide, see step 4.

Usually, the number of acidic and basic amino acids exceeds 50% of the total number of amino acids and can be solubilized with water.

The stability of peptide products is not only related to the sequence of the peptide, but also depends largely on the production process, and the stability of the products obtained by different processes varies greatly, especially the sequences containing amino acids such as Cys, Gln, Met, Asn and Trp.

Peptides that need to be stored for a long time should be stored in the form of lyophilized powder in sealed containers containing desiccant at -20°C, -80°C is better, which can avoid peptide degradation to the greatest extent possible. This storage allows the peptide to be preserved for several years, avoiding degradation and oxidation by bacteria, as well as avoiding the formation of secondary structures. If the sequence contains easily oxidized amino acids such as Cys and Met, it is recommended to store it at low temperature after filling with nitrogen.

Chemically synthesized peptides often carry free amino and free carboxyl groups. And the sequence of the peptide often represents the sequence of the parent protein. In order to be closer to the parent protein, the peptide terminus often needs to be closed, i.e., N-terminal acetylation and C-terminal amidation, and these modifications will reduce the total charge of the peptide, lower the solubility of the peptide, and also enable the peptide to mimic its original state of α-amino acid and carboxylic acid group in the parent protein.

If an acetylation modification of the N-terminus or an amidation modification of the C-terminus is required, please specify when placing your order. Once the synthesis is started, the C-terminal amidation cannot be modified; once the synthesis is finished, the N-terminal acetylation cannot be modified.

The difficulty of peptide production is directly related to its designed sequence, which can be predicted more accurately by our technicians. If the designed sequence is difficult to produce, it will not only affect the progress of the experiment, but also sometimes even if the product is produced, it will face various problems in use, especially the solubility problem.

Most peptides can be predicted to be soluble based on their sequences, some sequences are more difficult to determine their solubility and need to be confirmed experimentally. When you design the sequence, we can give you suggestions for smoother experiments.