Reconstituting a Peptide
- Bring frozen or refrigerated peptides to room temperature in a desiccated chamber to avoid water absorption.
- Always begin by reconstituting a small amount of peptide before committing the entire lot.
- Use sterile water or sterile filtration. If there are any Methionine (M), Cysteine (C), or Tryptophan (W) residues, use oxygen free solvents to prevent oxidation.
- Avoid reconstituting a peptide in a buffer, such as PBS. Salts hinder solubility.
- Choose the appropriate solvent. Begin reconstituting at a concentration higher than your desired final working concentration.
|
Amino Acid Characteristics |
Recommended Solvent |
|
Hydrophilic residues (KRHDEN) |
H2O |
|
Hydrophobic residues (AVLIMFW) |
Low solubility in aqueous solvents; are soluble in organic solvents (DMF, DMSO, TFA, Acetonitrile) |
- A solubilized peptide is completely clear. No flecks or cloudiness should be present.
- If a peptide with more hydrophilic residues is still not completely reconstituted:
- Adjust the pH of the solution according to the overall charge of the peptide
- Count the possible POSITIVE charges (K, R, H, and free N-terminus)
- Count the possible NEGATIVE charges (D, E, and free C-terminus)
- Determine which is greater.
- If positive charges are greater, add dilute acid dropwise to proteinate residues and maximize charge.
- If negative charges are greater, add dilute base dropwise to deproteinate and maximize charge.
- try sonication, gentle heat or an organic solvent, such as DMSO, Acetonitrile or DMF.