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PEPTIDE DISSOLVING METHOD


PEPTIDE DISSOLVING METHOD                                                            http://www.acinopeptide.com                                              

There is no ideal solvent that will solubilize all peptides, maintain their integrity and be compatible with biological assays. Thus, a series of increasingly powerful solvents may have to be tried until the peptide dissolves.

Step 1
I
n general, try to dissolve the peptide in sterile distilled water or sterile dilute acetic acid (0.1%) solution to give a stock solution at a higher concentration than required for the assays. The peptide can be diluted later with the assay’s buffer, but if the buffer is used initially and peptide fails to dissolve, recovery of the pure peptide free of nonvolatile salts and organic solvents can be difficult. If the peptide persists as visible particles, sonication can be tried. Sonication breaks down lumps of solid peptide to smaller particles and vigorously stirs the solution, improving the rate of dissolution, but does not alter the solubility of the peptide in the particular medium being tried. If, after sonication, the „solution“ has gelled, has a persistent haziness, or has a scum (not bubbles) floating on the surface, the peptide has probably not dissolved but is simply finely suspended.

Step 2
If the peptide was insoluble, look at the peptide sequence before proceeding. What proportion of the amino acid residues are hydrophobic (A,F,I,L,M,P,V,W,Y,C)? How many positive (K,R,H and N-terminus) and how many negative (D,E and C-terminus) charges are there? What is the overall (net) charge at neutral pH?If the sequence has a net charge at neutral pH, addition of dilute acetic acid as suggested above (for basic, positively charged peptides) or dilute aqueous ammonia or ammonium bicarbonate (for acidic, negatively charged peptides) with further sonication should improve solubility. The final concentration of acetic or ammonia/ammonium bicarbonate you use will depend on what assay system can tolerate. If the peptide still refuses to dissolve, you can at least remove the volatile buffer solution by lyophilisation and try alternative solvents on the same peptide sample.

Step 3
If the sequence has a little or net charge at any pH, and if the number of hydrophobic residues approaches 50% or more, the above procedures will probably be inadequate. Addition of acetonitrile, ethanol, dimethylformamide (DMF), dimehylsulfoxide (DMSO), or the use of chaotropic salts as guanide hydrochloride or urea, will dissolve most peptides.If it is known that the peptide is slightly soluble in aqueous solution, it is better to dissolve it completely an a small amount of neat acetic acid or DMF and slowly dilute with water rather than progressively adding such solvents to a suspension of the peptide in water.