How Do I Solubilize My Peptides?

As discussed above, before you order your peptide it is critical to note whether the peptide will be soluble in a medium that will be compatible with the experiments you wish to run. Often, when a competitor receives a peptide order, it will be made as instructed with no thought given to whether the customer thought about solubility issues. When we see a very hydrophobic peptide sequence, the customer will be called not only to discuss the difficulties in manufacturing the desired peptide, but also to insure that the customer will be able to utilize the peptide given the need for large amounts of organic solvents in order to make the peptide soluble. Normally, however, many peptides are easily solubilized as long as you keep the primary amino acid sequence in mind, and follow the guideline below. We recommend that you first attempt to solubilize a small portion of the peptide to insure that all of the peptide is not put into an inappropriate solvent. In some cases, this might require the peptide to be repurified due the presence of salts. For all peptides, the Certificate of Analysis will indicate a preferred method of solubilization, however after the final lyophilization and dependent upon your particular requirements, you may find it necessary to take other action to solubilize your peptide.

  1. 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).
  2. 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).
  3. Add up the charge, thus determining the overall charge of the peptide.
  4. For peptides with an overall charge of 1 or higher (a basic peptide) first try to dissolve the peptide in water. 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 solubilize peptides but it is not cell-friendly and thus is used only if acetic acid fails to help solubilize the peptide. After the addition of TFA, the peptide should be diluted to approximately 1ml with deionized water. If the peptide still failed to solubilize and your experiments are not compatible with the use of organic solvents (see 6 below), you should consult 21st Century Biochemicals.
  5. For peptides with an overall charge of less than zero (acidic peptide) first try to dissolve the peptide in water. If the peptide does not dissolve, add ammonium hydroxide (NH4OH 10-50ul) and once in solution, dilute the peptide to approximately 1ml with deionized water. If the peptide still failed to solubilize and your experiments are not compatible with the use of organic solvents (see 6 below), you should consult 21st Century Biochemicals. Note: Caution must be used, however, with peptides that contain cysteine (C), as the used of alkaline pH can cause disulfide bond formation.
  6. For peptides with an overall charge of zero (neutral peptide), solubilization may require the addition of various organic solvents, such as acetonitrile, DMSO, methanol, propanol or isopropanol. Denaturing salts, such as urea or guanidinium-HCL are used as a last resort.
    Examples of peptides and their recommended solubilization methods:
    • VSRLGGKSIEVKIMPL [+4] + [-2] = +2 A basic peptide – see method #4 above.
    • acetyl-VSRLGGKSIEVKIMPL-amide [+3)]+ [-1] = +2 A basic peptide – see method #4 above.
    • 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.



 

How Do I Solubilize My Peptides?
What Amino Acids Will Cause Problems in the Synthesis of My Peptide?
What Purity is Right for My Peptide?
How Do I Choose a Peptide Sequence?
How Difficult is it to Synthesize a Custom Peptide?

 

 

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