1.The main problem of peptide solubilization is the formation of secondary structure. Although the formation of the secondary structure of the hydrophobic peptide chain is more obvious, this phenomenon occurs in almost all peptide chains except for the shortest peptide chain, regardless of polarity. Therefore, the first principle for dissolving peptides is to use sterile distilled water or deionized water, of course, oxygen-free water is best. The peptide solution may be subject to bacterial degradation. To prevent this from happening, it should be dissolved in sterile distilled water or filtered with a 0.45 or 0.2 pore size filter. Peptides containing Cys, Met, and Trp are easily oxidized and should be dissolved in oxygen-free water. Oxygen-free water can be obtained by injecting inert gas (nitrogen, helium, argon) and degassing under reduced pressure.
2. If the peptide is not soluble in pure water, ultrasonic treatment can help break up the particles and increase solubility. Note: Ultrasonic treatment can cause heating of the solution or degradation of peptides.
3. If the peptide contains multiple basic amino acids, use (1-10%) aqueous acetic acid: For peptides with strong hydrophobicity, use 50% acetic acid.
4. If the peptide contains a lot of acidic amino acids, it can be dissolved in ammonia solution (1-10%) or volatile alkaline buffer such as ethylmorphine acetate or bicarbonate. The pH value must be adjusted before chromatography.
5. Isopropanol and acetonitrile can dissolve medium-sized peptides. If the peptide is to be loaded onto the column, the amount of organic solvent must be small or it will seriously affect the residence time.
6. If the polypeptide contains Val, Leu, Met, Phe, Tyr, Ala and other aromatic chains and is highly hydrophobic, or is a neutral peptide, the use of mold denaturants such as DMF or DMSO will facilitate the dissolution of the polypeptide.
a. High-concentration mold denaturants help dissolve by destroying the secondary structure of the polypeptide.
b. Membrane denaturant is suitable for the preparation of peptide analysis solution, but may interfere with the research of its biological activity.
c. DMF is the best denaturant (the highest concentration can reach 30%), and it is added dropwise until the peptide is dissolved.
d. In reversed-phase chromatography, DMF will flow out together with the front of the eluent. Depending on the amount of input, the peak value may be very high. Most peptides can flow out within a few minutes after a large amount of DMF flows out. If the peptide chain is small and the elution is too early, the amount of peptide will be very low.