Custom Peptide Solubility

Preparing Custom Peptide Solution

How should a peptide solution be prepared or how do I reconstitute my peptide?

Since there is not a universal solvent for dissolving every peptide, this crucial step is not always straightforward as it may appear.  Because several solvent systems may be necessary until the desired conditions are achieved, always test a small sample of the peptide to determine the best solvents for complete solubilization.  When testing, begin with solvents that can be easily removed by lyophilization, such as water and acetic acid.  For this reason, it is not recommended to start with buffers which have high salt concentrations.  Vivitide also offers solubility testing as an additional service for customers, should you desire it.

• Bring frozen or refrigerated peptides gradually 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 higher concentration than your desired final working concentration.

A completely solubilized peptide is a clear solution. 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 protonate residues and maximize charge.
• If negative charges are greater, add dilute base dropwise to deprotonate and maximize charge.
• Try sonication, gentle heat or an organic solvent, such as DMSO, acetonitrile or DMF.

If your peptide is still not completely reconstituted, re-lyophilize the peptide and begin again.

Please contact Vivitide Technical Service for assistance, should you have further difficulty.

Determine the overall charge

Charged amino acids aid solubility in aqueous environments.  A sequence with little or no overall charge at any pH is not likely to be water soluble.

    Hydrophobic amino acids:  Ala, Phe, Ile, Leu, Val, Pro, Met, Trp, Tyr, Cys

    Positive Charges:  Lys, Arg, His and Free N-terminus

    Negative Charges: Asp and Glu and Free C-terminus

First, determine if the peptide is charged or neutral by calculating the overall charge of the peptide at pH 7.  Calculate overall charge by using the following values:

    +1 for each basic residue (Lys, Arg, and N-terminus)

    -1 for each acidic residue (Asp, Glu and C-terminus)

    For His, use +1 at pH 6

If the overall charge of the peptides is negative then the peptide is acidic, if it is positive then it is basic, and if zero then it is neutral.

Charged peptides

For acidic peptides (and/or if the total number of charges of the peptide at pH 7 is greater than 25% of the total number of residues): use a small amount of 0.1M ammonium bicarbonate to dissolve the peptide, and then dilute it with water to the desired concentration.  Maintain pH around 7 and adjust pH as needed.  For basic peptides (and/or if the total number of charges of the peptide at pH 7 is between 10-25% of the total number of residues): use a small amount of 25% acetic acid to dissolve the peptide and dilute it with water to the desired concentration.  Adjust pH with 8 M NH4OH to desired pH for oxidation (4-7).  For neutral peptides (and/or if the number of charges is greater than 25% of the total number of residues): use the strategy described for acidic peptides.  Otherwise, the use of organic solvents is recommended.

Hydrophobic or neutral peptides

Hydrophobic peptides containing 50% to 75% hydrophobic residues may be insoluble or only partially soluble in aqueous solutions, even if the sequence contains 25% charged residues.  It is best to first dissolve these peptides in a minimal amount of stronger solvents such as acetonitrile, isopropyl alcohol, ethanol, and/or acetic acid, and then slowly add (drop wise) the solution to a stirred aqueous buffer solution.  If the resulting peptide solution begins to

show turbidity, you might have reached the solubility limit and it will be futile to proceed.  Again, it is important to remember that the initial solvent of choice

should be compatible with the experiment.  It is important to dissolve the peptide completely in the initial solvent (such as acetic acid, acetonitrile) because

the rate of dissolution of peptides into these solvents is usually higher than in a water/solvent mixture.  If a water/solvent mixture is used first to dissolve the peptide, the final volume of solution may be larger than necessary.

Sonication

It may be necessary to sonicate the solution before determining if the solvent choice was appropriate.  Sonication should improve solubilization by breaking the solid peptide into smaller particles.  If the solution gels, becomes cloudy or turbid, or has visible particulates, the peptide has not dissolved completely but is suspended.  At this point, a stronger solvent is necessary and the solvents should be removed.  Begin again with the dry sample.

Why does my KLH/peptide solution appear cloudy?

KLH (Keyhole Limpet Hemocyanin) is a large (MW = 4x105 - 1x107 kDa) aggregating protein. Because of its size and structure, its solubility in water is often limited. This can manifest in tendrils or a general cloudy appearance. This does not affect antigenicity, and the turbid solution can be used for immunizations.

Liability Disclaimer

All products sold by Vivitide are intended solely for laboratory and research use and should not be used in or on human subjects.  User assumes all risk of patent infringement by reason of use of material provided by Vivitide. Vivitide will not be responsible for damages arising from misuse of any product and is not responsible for the results of research using our products.