Start with an appropriate choice of solvent that is both compatible with yourexperimental procedures and that does not react with or degrade the peptide.The solvent should be dry and degassed. Since most peptide work is small-scale, it is most practical to buysmall volumes of dry solvent, rather than attempting to dry it yourself.  Determine whether the peptide is acidic,basic or neutral and proceed with solubilization using a small amount of thepeptide. Acidic and basic peptides are more soluble at neutral pH than acidicpH.

Peptides are acidic when the manufacturing process is complete, due to the presence oftrifluoroacetate (TFA) as the counter ion (a result of the cleavage orpurification process). Check whether the peptide is supplied as a TFA saltbefore you start the purification process.

Short non-hydrophobic peptides (less than 5 amino acids) and peptides containing>25% non-clustered, charged residues and <25% hydrophobic residues willtypically dissolve in aqueous solutions.

Simply adding water may dissolve basic peptides. If it does not, first try a drop(10-20 µl) of glacial acetic acid and sonicate or vortex. This may be increasedup to 30% acetic acid by volume for problematic sequences. Addition of base canalso promote oxidation of cysteines to the disulphide so deoxygenated buffersshould always be used.  For basicpeptides with net charge of +1 or greater, an acidic solution will be needed.

Acidic peptides with net charge of -1 or greater should be dissolved in a small amountof basic solvent such as 0.1% ammonium hydroxide or ammonium bicarbonate anddiluted to the required stock concentration with water. The exception ispeptides containing Cys, as disulphide bonds may form at alkaline pH.

Neutralor hydrophobic peptides with a net charge of zero can sometimes be brought intosolution by addition of base, but often a gel forms. Generally this gel willonly respond to dilution with higher amounts of distilled, deionized water,along with sonication and vortexing.

Peptides that are >50% hydrophobic may be difficult to dissolve in water alone andshould be dissolved in a small amount of organic solvent, for exampleacetonitrile, methanol, isopropanol, dimethyl suphoxide (DMSO),dimethylformamide (DMF). This should be added drop wise, followed by sonicationand vortexing after every drop until the peptide dissolves. The drop wiseaddition of the organic solvent can also be used for peptides that do notrespond to pH adjustment.  Note that somecell culture based assays may not react well to DMSO, so a different solventshould be considered.

Peptides that are >75% hydrophobic are unlikely to dissolve in aqueous solution aloneand may require solubilization in a stronger solvent such as TFA or formic acidand at high concentration. The peptide may precipitate out when aqueous bufferis added. These conditions may not be compatible with some cell culture basedexperiments.

Organic solvents at certain concentrations are incompatible with some biochemicalassays. A small amount of DMSO should be compatible with most immunologicalassays, but avoid DMSO if the peptide contains Methionine, Cysteine orTryptophan due to sulphoxide or disulphide formation.   These peptides should be prepared using1,2-ethanediol (EDT) or dithiothreitol (DTT) in order to preventoxidation.  Oxygen-free water or buffers,or DTT are recommended for solubilizaton.Note, if DMSO solvent is at all wet itwill not freeze at -20°C, so freezing at -30°C is recommended.  DMSO is hygroscopic and will adsorb water ifsubjected to repeated freeze-thaw cycles. To minimize these issues, buy small volumes of dry DMSO forsolubilzation purposes.

Peptidesthat are prone to aggregation may require strong denaturants (e.g., 6M urea orguanidine hydrochloride), which may then be able to be diluted.

If peptides are to be used in cellular assays, start by making a concentratedstock solution e.g., 5-10 mM, or 5-10mg/ml. Dilute the peptides into physiological buffer for use, such that theoriginal solvent is present at no more that 0.1% in the final workingsolution.  Diluted peptide solutions mayalso be filter-sterilized (0.22mm) if they are to be added to sterile cultures.