How Do I Solubilize My Peptides?

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. This is why all peptide requests are an-

alyzed by our staff and you may be contacted by our scientific staff to discuss peptide request where

solubility or other factors may be an issue. 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.

1.

Assign a value of -1 to each acidic residue that are Asp (D) and Glu (E) as well as the C-termi-

nal –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 pep-

tide 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 di-

luted 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 con-

sult 21st Century Biochemicals.

5.

For peptides with an overall charge of less than zero (acidic peptide) first try to dissolve the pep-

tide 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 addi-

tion 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 pep-

tides 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 pres-

ence of cysteine, caution should be used. Thus water or a small amount of organic

should be tried first.