The amino acid composition of the peptide affects the ease of solubility.
The properties of different amino acids contribute to the overall charge, molecular weight and hydropathy of a peptide.
When designing peptides it is advisable to have at least 20% charged residues to aid solubilization. See Peptide Design.
Recommendations for solubilization of individual custom peptides
Start with an appropriate choice of solvent that is both compatible with your experimental 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 buy small 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 the peptide. Acidic and basic peptides are more soluble at neutral pH than acidic pH.
Peptides are acidic when the manufacturing process is complete, due to the presence of trifluoroacetate (TFA) as the counter ion (a result of the cleavage or purification process). Check whether the peptide is supplied as a TFA salt before 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 will typically 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 increased up to 30% acetic acid by volume for problematic sequences. Addition of base can also promote oxidation of cysteines to the disulphide so deoxygenated buffers should always be used. For basic peptides 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 amount of basic solvent such as 0.1% ammonium hydroxide or ammonium bicarbonate and diluted to the required stock concentration with water. The exception is peptides containing Cys, as disulphide bonds may form at alkaline pH.
Neutral or hydrophobic peptides with a net charge of zero can sometimes be brought into solution by addition of base, but often a gel forms. Generally this gel will only 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 and should be dissolved in a small amount of organic solvent, for example acetonitrile, methanol, isopropanol, dimethyl suphoxide (DMSO), dimethylformamide (DMF). This should be added drop wise, followed by sonication and vortexing after every drop until the peptide dissolves. The drop wise addition of the organic solvent can also be used for peptides that do not respond to pH adjustment. Note that some cell culture based assays may not react well to DMSO, so a different solvent should be considered.
Peptides that are >75% hydrophobic are unlikely to dissolve in aqueous solution alone and may require solubilization in a stronger solvent such as TFA or formic acid and at high concentration. The peptide may precipitate out when aqueous buffer is added. These conditions may not be compatible with some cell culture based experiments.
Organic solvents at certain concentrations are incompatible with some biochemical assays. A small amount of DMSO should be compatible with most immunological assays, but avoid DMSO if the peptide contains Methionine, Cysteine or Tryptophan due to sulphoxide or disulphide formation. These peptides should be prepared using 1,2-ethanediol (EDT) or dithiothreitol (DTT) in order to prevent oxidation. Oxygen-free water or buffers, or DTT are recommended for solubilizaton.Note, if DMSO solvent is at all wet it will not freeze at -20°C, so freezing at -30°C is recommended. DMSO is hygroscopic and will adsorb water if subjected to repeated freeze-thaw cycles. To minimize these issues, buy small volumes of dry DMSO for solubilzation purposes.
Peptides that are prone to aggregation may require strong denaturants (e.g., 6M urea or guanidine hydrochloride), which may then be able to be diluted.
If peptides are to be used in cellular assays, start by making a concentrated stock solution e.g., 5-10 mM, or 5-10mg/ml. Dilute the peptides into physiological buffer for use, such that the original solvent is present at no more that 0.1% in the final working solution. Diluted peptide solutions may also be filter-sterilized (0.22mm) if they are to be added to sterile cultures.
Recommendations for dissolving Prospector Custom Peptide Libraries
Complete solubilization of peptides is important for successful screening of peptide activities. Peptides can be fully active only if they are completely solubilized and are able to assume the correct conformation for binding to their receptors. As the number of peptides in a set increases, so does the potential solubility variation of the peptides within the set. Therefore, in order to obtain accurate and reliable peptide activity data, careful attention should be devoted to the process of dissolving peptide sets.
The strategy for dissolving the PEPscreenŽ peptide set, or any of the other Prospector peptide libraries, is different from dissolving individual peptides. For individual peptides, conditions are chosen for optimum solubility based on the given peptide sequence. However, for peptide sets, conditions are chosen in an effort to dissolve as many of the peptides in the set as possible in the first solubilization attempt. Suggested common strategies are schematically represented below:
Full details of the steps above: Guidelines for dissolving peptide sets
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