Peptide Handling, dissolution & Storage

The following data was supplied with the peptides by the manufacturer;

• Safety
• Storage
• Container
• Dissolution
• Dissolution approach 1
• Dissolution approach 2
• Dissolution approach 3
• Storage of Solutions
• Feedback notes

Material Safety Note: THIS PRODUCT IS NOT LICENSED OR APPROVED FOR ADMINISTRATION TO HUMANS OR ANIMALS

Use due caution in handling and use of this product. A Material Safety Data Sheet for this investigational material is not available as its chemical, physical and toxicological properties have not been fully investigated. Exercise due care. Do not take internally. Avoid breathing dust. Wear suitable protective clothing and gloves. If any irritation occurs, obtain immediate medical attention.
Do not release spilled or wasted material to the environment. This material should be handled in a restricted manner until its degree of risk has been adequately assessed.
NIBSC CJD Resource Centre is not aware of any specific hazard data applicable to this material nor of any requirements regarding the restricted disposal of the compound. Standard Laboratory Procedures should be employed when handling this material. If in doubt contact your Site Safety personnel for advice.
NIBSC CJD Resource Centre cannot guarantee completeness or accuracy of the information contained herein and further disclaims all liability for its handling or use.

Storage of peptides:

Peptides should be stored in a dry, cool, dark place. For best preservation, store at 4°C or colder away from bright light. Dry peptides are stable at room temperature for days to weeks but for long-term storage -20°C is to be preferred.
Contamination with moisture will greatly decrease long term stability of solid peptides. A vial containing a peptide should be allowed to warm to room temperature prior to being opened. After removing the desired quantity, the vial should be re-sealed, preferably under an atmosphere of dry inert gas, and then returned to cold storage.

Choice of containerAn ideal container for peptide manipulation should be clean, chemically inert, optically clear, strong and available in an appropriate size. Glass and plastic vials are generally satisfactory for the purpose, however, care does need to be taken with plastic vials when organic solvents are to be used. Polypropylene vials are both strong and chemically inert but if high visibility is required glass is a better option. Please appreciate that peptides in solution can and do adsorb to many materials. This may occur to varying extents being dependent upon factors such as constituent amino acids, vial material, and peptide concentration. At high dilution it is possible to lose large percentages of peptide due to adsorption to surfaces thereby grossly distorting subsequent results. Use of high quality specialist glass and polypropylene vials can lessen this problem.

Peptide dissolutionThere is no ideal solvent that will solubilise all peptides whilst maintaining their integrity and being compatible with biological assays. It may prove necessary to use a series of increasingly powerful solvents until the peptide dissolves. For peptides with a solubility problem the following guidelines may prove of help.

Approach 1:In general, attempt to dissolve peptides in sterile distilled water or sterile dilute acetic acid (0.1%) solution to give a stock solution at a higher concentration than that required for subsequent use. This solution may later be diluted with an appropriate buffer. If the peptide persists as visible particles, sonication may prove of help as it improves the rate of dissolution. If, after sonication, the ’solution’ has gelled, has a persistent haziness, or has a scum floating on the surface, the peptide has probably not dissolved but is simply finely suspended.

Approach 2:If the peptide remains Insoluble, look at Its amino acid composition prior to proceeding further. What proportion of amino acids are hydrophobic (A, C, F, I, L, M, P, V, W, Y) and how many residues are positively charged (K, R, H, and amino terminus) or negatively charged (D, E, and carboxy terminus)? What is the overall net charge at neutral pH? If there is a net charge at neutral pH, addition of dilute acetic acid (for basic, positively charged peptides) or dilute aqueous ammonia or ammonium bicarbonate (for acidic, negatively charged peptides) with further sonication should greatly aid solubility. The final concentration of acetic acid or ammonia/ammonium bicarbonate allowable will be determined by the use to which the peptide is to be finally put. If the peptide still refuses to dissolve, these volatile buffer systems may be removed by lyophilisation and alternative solvents tried on the same peptide sample.

Approach 3:If the peptide sequence has little or no net charge at any pH, or if the number of hydrophobic residues approaches 50% or more, +he chances are +ha+ +he above procedures may prove inadequate. Addition of acetonitrile, ethanol, dimethylformamide (DMF) dimethyl sulphoxide (DMSO), or the use of chaotropic salts such as guanidine hydrochloride or urea should aid the dissolution of most peptides. The choice will depend largely on the compatibility of such reagents with the subsequent peptide application. If it is known that the peptide is slightly soluble in aqueous solution, it is better to first dissolve it completely in a small amount of neat acetic acid or DMF and then slowly dilute with water or buffer rather than to add such solvent progressively to a suspension of the peptide in aqueous systems

Storage of peptide solutions:The shelf life of peptide solutions is limited especially for peptides containing C, M, N, Q, and W. To prolong the storage life of peptides in solution, sterile buffers with a pH of around 5-6 should be used. Aliquots should be stored at -20°C or colder wherever possible. Avoid the use of frost-free freezers, which vary enormously in temperature during the frequent automatic defrosting cycles. Repeated freeze-thaw cycles are deleterious to peptides.

Feedback Note:

28/08/03

-They all seemed to dissolve ok in DEPC treated water with the exception of NPOA0/0116 ’H-ERVVEQMCITQYERESQAYY-OH’ This needed a small amount of DMSO first and then further diluted in water