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Peptide Solubilization Handling Guideline

Proper peptide handling and solubilization is the starting point of a successful bioassay project . If reconstitution is a big concern for you,Use a small aliquot of peptide to test the reconstitution conditions: 

Once satisfied, apply to the larger aliquot as needed.  You may request your custom peptide synthesis to be delivered in several aliquots at the time of ordering.

The initial solvent used should be the most appropriate one:

For example, for a very hydrophobic peptide, it is recommended to dissolve it first in a small volume of organic solvent (such as DMSO or acetonitrile) before applying the aqueous solution. Adding organic solvent to a suspension of hydrophobic peptide in aqueous solution is not likely to help in dissolving. In other words, if an aqueous buffer is being added first to a hydrophobic peptide powder, adding organic solvent afterwards would not likely help much in dissolving the peptide well. 

Determine the initial solvent:

For a short peptide which is 5aa or less - try sterile distilled water and it is likely to dissolve.

For a longer peptide (>5aa), the overall charge of the peptide will determine which initial solvent to use: 

Assign a value of  -1 to acidic residues: Asp(D), Glu(E), and the C-terminal free acid(-COOH);

Assign a value of +1 to basic residues: Arg (R), Lys (K), His (H), and the N-terminal free amine(-NH2);

Then, calculate the overall charge of the entire peptide:

1.  If the overall charge of the peptide is positive (a basic peptide), try to dissolve the  peptide in sterile distilled water first. If water fails, add ~20% acetic acid solution. If the peptide still does not dissolve, add drops of TFA (< 50ul), or use 0.1%TFA/H2O to solubilize the peptide. Then dilute the peptide solution to the desired concentration. 

2.  If the overall charge of the peptide is negative (an acidic peptide), try to dissolve the peptide in sterile distilled water first. If the peptide persists as visible particles, sonication can be tried. If water fails, add NH4OH (<50ul)or 0.1%NH4OH drop-wise. Then dilute the peptide solution to the desired concentration. If the peptide contains Cys, do NOT use basic solutions (NH4OH); use DMF instead. 

3.  Peptide whose overall charge is 0 (a neutral peptide). It usually dissolves in organic solvents, such as acetonitrile, methanol, or isopropanol. If this does not dissolve completely:

a) For peptides that tend to aggregate (due to the hydrophobic interaction), the addition of denaturants, such as 8M urea or 6M guanidine-HCl, may also be required. 

b) For very hydrophobic peptides (containing more than 75% hydrophobic residues), add DMSO drop-wise (use DMF instead for Cys containing peptides), and then dilute the solution with water to the desired concentration.

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