Good morning, Mr. Ashby

Organic solvents are out too? dimethylformamide? PEG aqueous mixtures?

Best regards.

You might want to include 1 mM DTT (fresh-prepared). It will help to keep all your Cys residues in reduced conditions.

Hi Gustavo ,  some level of organic solvent should be fine as long as I can dilute it down in water. For example if I can dissolve at 1mg/ml then the concentration of organic solvent can be around 5% as I will do another 1:10 dilution in water before using it in the assay (and I think 0.5% should be fine). Is there a particular solvent then seems to works best for you?

Hi Viktor, thanks for your answer, I did try some DTT but at a lower concentration and it had no effect. At what time should you add the DTT? after the peptide has been mixed with water ?

@Martin

Your cysteine-containing peptides may have a high propensity to oligomerize by forming disulfide bridges. You may consider using TCEP (tris(2-carboxyethyl)phosphine) as a biocompatible, water-soluble, and inexpensive reducing agent to prevent peptide oligomerization. Here are analogous examples of this approach in live-cell assays (both open-access, see Supplementary Information for reagent and protocol details):

• From researchers at the National Institutes of Health (NIH, USA):

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3307366/

• From researchers at Umeå University (Sweden)

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4030451/

Hope this helps. 

I would suggest to dissolve your peptide in 100 mM DTT immediately after synthesis (to make sure that all disulphide bonds are reduced), then desalt it in the buffer containing 1 mM DTT (to keep all Cys residues in reduced conditions). Treatment of the peptide with 1 mM DTT immediately after synthesis will not reduce all disulphide bonds. All buffers should have pH 7.5 or higher.

Hi Ashby,

I have also faced the same problem for 16mer peptide containing 2 cysteine residues.

My peptide contains 4 Lys residues, so i could able to dissolve it in 30% AcOH/Water. 

AcOH is best solvent for positively charged peptides. You can try this.

Of course, there will be a problem based on sequence of the peptide. For example before disulfide formation they have good solubility however after disulfide formation they leads to aggregation because of the conformational changes. This was unable to resolved in my case. 

Please any body can give me suggestion in this regard.

Thanks in advance.

All the best.

Also keep your reduced peptides under inert atmosphere

dear sir

try in THF and DMAP

because DMAP is a good peptide agent and it is basic in nature.

Martin,

if you have problems with 100% reduced form of your peptide,  you definitely have to use organic solvents. I'd solve it in acetonitrile and then add water/buffer. Just keep all solvents degassed (you know how to do it, do you?).

Hi Martin .

I suggested PEG because to you is, tipically, free of biological activity and very versatile in preparation of aqueous solutions. PEG is one of the most (if not the most) widely used cosolvence agent in pharmacy.

Now, if organic solvents don´t affect the active conformation of youre peptide you can use the solvent as blank in the eventuality that you can´t achieve satisfactory dissolution. But I think that PEG will work.

Regards. 

Try dissolving it in DMSO, then dilute with water until you get the concentration you want for the bioassay. Once the peptide is fully dissolved, it is quite hard for it to crash out after you add water

DMSO or DMF would be a good choice but you have to consider to use it from 5 to 10% of the final concentration. It is commonly used for biological studies.

for biological  study has to dissolve either wateror buffer or finally DMSO

I will too suggest you use DMSO and then dilute in water or in the buffer you are going to use for your assays.

Do you need your Cys-containing peptides to be in the reduced form?

This a great discussion! But do you think that using DMSO is preferable for Cys-containing peptides? Disulfide bond or sulphide oxides may be formed?

What is the PI for your peptide! Be sure that your peptide doesn`t form disulfide bond! I`d dissolve in acidic pH around 6.

Bubble some H2S in your mixture before trying to disolve it.  And I would recommend

using DMF/CH2Cl2 but not DMSO

While considering dissolving cysteine polypeptides, it may be worth to consider the somewhat analogous case of keratin extraction. Keratin has significant cysteine content, being hence strengthened by thermally stable disulphide (-S-S-) bonds. Due to extensive disulphide bonding, keratin is generally difficult to dissolve without the assistance of reducing agents (e.g. sodium sulphide) or of keratinase enzymes able to promote proteolytic attack to disulphide bonds. A hydrophobic ionic liquid was alternatively proposed: Yun-Xian Wang, Xue-Jun Cao, "Extracting keratin from chicken feathers by using a hydrophobic ionic liquid", Process Biochemistry, 47(5) 2012, 896-899: http://www.sciencedirect.com/science/article/pii/S1359511312000888

Friends, High pH is needed to dissolve cystein. It is easy dissolved in NaOH (40%) by gently heating the tube.