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I have working on the realisation of on assay, that is intended to examine the activity of a protein. The assay works in a way, in which the product of the target enzyme is transfered by a support protein to a peptide. The peptide-product complex that is formed after the secondary reaction becomes fluorescently active and this is my activity/inhibition readout.

The unpolar peptide has a cysteine residue and therefore a DTT reduction must take place beforehand. Concentration of the peptide in the assay is 2 micromolar.

My plan was to create a peptide stock solution 200 fold concentrated to weigh-in the peptide more accurately (3mg on 10 ml buffer) and than dilute 1 part of the concentrated stock with 199 parts of the buffer to give the final concentration. Unfortunately, it was not possible to bring the peptide in solution in the stock. I tried warming it a little, vortexing and ultrasonic treatment. Adding 10 percent of DMSO to the stock did not clear the precipitate.

The peptide seams to be soluble in DMSO, but adding the peptide in DMSO to the buffer seems to result also in precipitation..... Can you give me additional Tipps? Would it be advisable to do the DTT treatment directly in DMSO and than try to get this into aquous solution? O would assume, that after the SS double bonds have cleaved, solubility should go up?

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    $\begingroup$ I maybe could try the following: Dissolve 3 mg in 1 ml of DMF, add the DTT and incubate. Than transfer to the buffer slowly, and hope that at this step no precipitation occurs. DTT is maybe better suited than DMSO because i of the cysteine? Final DMF concentration in the assay would be fairly low, under 0,1 % if the concentrations will work. $\endgroup$
    – raptorlane
    Commented Jan 7 at 15:37
  • $\begingroup$ Aside from solubility issues related to crosslinking, have you checked the solubility of a cysteine free peptide? (eg C->S). Can you use detergents? What data do you have on predicted solubility? $\endgroup$
    – Buck Thorn ♦
    Commented Jan 8 at 10:52
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    $\begingroup$ So, it is a very unpolar peptide, and it did not go into the buffer solution. What I tried, and what finally worked was using pure DMF as a solvent. To this I added the DTT and did the reduction entirely the organic solvent. Than I could transfer to the buffer without precipitation. $\endgroup$
    – raptorlane
    Commented Jan 11 at 17:37
  • $\begingroup$ Dealing with insoluble peptides is a pain. If you have some time you can write up what you did into an answer. Ideally you might provide more information about the length, predicted solubility or any other properties of the peptide to make the answer more useful (you might not want to provide the sequence if its important to keep under wraps). $\endgroup$
    – Buck Thorn ♦
    Commented Jan 11 at 18:07

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So, it is a very unpolar peptide, and it did not go into the buffer solution, neither vortexing, ultrasonic treatment or gentle heating worked. The 3 mg of peptide was attempted to be solved with 10 ml of buffer and a tensid was already included. What I tried, and what finally worked was using pure DMF as a solvent to create a stock solution. To this I added the DTT and I performed the reduction entirely in the organic solvent. Than I could transfer to the buffer in a 1:2000 ratio without precipitation, resulting in a concentration of roughly 5 micromolar. The assay system worked than as expected. When you are facing a similar problem, try to use DMSO to bring the peptide in solution. If the peptide contains cysteine, I would prefer DMF, because DMSO can oxidize cysteine and I don't know if DTT can fully compensate for this. DMSO is used up to 5 % in cell assays, but DMF is cytotoxic and might only be applicable for protein based assays. Other possible solvents might be acetic acid, acetonitrile, methanol, propanol, or isopropanol, pure or in mixture with water or buffer. If your peptide is acidic or basic instead try to use the corrosponding aqueous conditions first (PBS, acetic acid).

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