Bio Basic can offer free solubility testing to make recommendations on dissolving your peptide.
Price: FREE with peptide synthesis.
Turnaround: Varies with project.
Peptide solubilization can often be quite a tricky problem. For the first attempt to determine the solubility, we recommend dissolving an aliquot into your buffer of choice, by vortexing and sonication if necessary.
It is vitally important that before you order your peptide, determine if it will be soluble in a medium that will be compatible with the experiments you wish to run. After which, we recommend that you first attempt to solubilize a small portion of the peptide. This ensures that NOT all peptide is put into an inappropriate solvent.
For all peptides, usually water or diluted PBS solubilizes the peptide, however after final lyophilization and dependent upon your particular requirements, you may find it necessary to take other actions to solubilize your peptide. Normally, many peptides are easily solubilized as long as you keep the primary amino acid sequence in mind, and follow the guidelines below:
Assign a value of "-1" to each acidic residue that are Asp (D) and Glu (E) as well as the C-terminal –COOH (not present if peptide is made as an amide).
Assign a value of "+1" to each basic residue that are Arg (R), Lys (K), His (H), and the N-terminal - NH2 (not present if the peptide was acetylated).
Add up the charges, thus determining the overall charge of the peptide.
For peptides with an overall charge of 1 or higher (basic peptides), first try to dissolve the peptide in water or 1X PBS.
Alternatively, add a drop or more of glacial acetic acid to facilitate the dissolution (can be up to 20-30% in volume for problematic sequences).
e.g: add 0.9mL ddH2O and THEN 0,1ml of 10XPBS.
If the peptide does not dissolve, add 10% acetic acid dropwise with vortexing in between. The peptide solution can also be warmed slightly. Longer peptides (20+ amino acids) with a small overall net charge might require the addition of a stronger acid. Trifluoroacetic acid (TFA 10-50ul) is often used to help peptides soluibilization but it is not cell-friendly. After the addition of TFA, the peptide should be diluted to approximately 1ml with deionized water so that final concentration is less than 1%.
For peptides with an overall charge of less than zero (acidic peptides) first try to dissolve the peptide in water or 1X PBS. If the peptide does not dissolve, add ammonium hydroxide (NH4OH 10-50ul) and once in solution, dilute the peptide to approximately 1ml.
(5% ammonia may be helpful for solubilization).
With deionized water.
Note: Caution must be used, however, with peptides that contain cysteine (C), as the use of alkaline pH can cause disulfide bond formation.
If the peptide still fails to solubilize, please try some organic solvent e.g. DMSO.
For peptides with an overall charge of zero (neutral peptides), solubilization may require the addition of various organic solvents, such as acetonitrile, DMSO, methanol, propanol or isopropanol. Denaturing salts, such as urea or guanidium-HCL are used as a last resort.
e.g: Add sequentially 0.1ml of DMSO (make sure peptide dissolves at this stage) + 1.7ml ddH2O + 0.1ml of 10XPBS (keep 5% DMSO).
Examples of peptides and their recommended solubilization methods:
a) VSRLGGKSIEVKIMPL [+4] + [-2] = +2 A basic peptide – see method #4 above.
b) acetyl-VSRLGGKSIEVKIMPL-amide [+3] + [-1] = +2 A basic peptide – see method #4 above.
c) acetyl-CGDLVGIKRETEYPRLAV [+3] + [-4] = -1 An acidic peptide but given the presence of cysteine, caution should be used. Thus water or a small amount of organic should be tried first.
For difficult hydrophobic peptides (containing high portion of A, V, M, I, Q where the net charge may or
may not be 0), try to first dissolve it in an organic solvent such as DMSO and ethanol, etc. or an
organic/aqueous co-solvent, then dilute the stock to the concentration and media of choice.
Note: The use of base and DMSO may promote problems for peptides containing cysteine residues with free thiol groups.