DMSO Stock Solutions: Preparing, Storing and Diluting Peptides for Assays

When DMSO is used

Dimethyl sulfoxide (DMSO) is a polar aprotic solvent that dissolves a wide range of polar and nonpolar molecules and mixes with water in all proportions. It is the default solvent for compound libraries and for peptides too hydrophobic to dissolve in water at stock concentrations.

DMSO is not the first choice for every peptide. Many sequences dissolve in water or in dilute acid or base chosen from the net charge (see solubility and aggregation). When DMSO is used, it acts as a cosolvent in the final assay and has to be treated as part of the experiment.

Preparing a molar stock

Assay stocks are usually made at a round molar concentration such as 10 mM so that later dilutions are simple. The mass of peptide, not the mass of powder, sets the molar amount, so the net peptide content matters.

A worked example: 2.00 mg of MOTS-c powder (molecular weight 2174.5 g/mol) with an assumed net peptide content of 80% contains 1.60 mg of peptide, or 1.60 / 2174.5 = 0.736 µmol. For a 10 mM stock that needs 0.736 µmol / 10 mM = 73.6 µL of DMSO. If the powder were treated as pure peptide, the calculation would call for 92.0 µL and the stock would actually be 8.0 mM, 20% below its label. The molarity calculator does the mass-to-molar step.

DMSO has a density of about 1.10 g/mL and is more viscous than water, so air-displacement pipettes can deliver slightly different volumes than they do with water. Weighing the solvent is a simple check: 73.6 µL of DMSO should weigh about 81 mg.

Water uptake, freezing and storage

DMSO is strongly hygroscopic: an open tube or plate takes up water from the air. Water dilutes the stock, since 10 µL of absorbed water in 100 µL of a 10 mM stock lowers it to 9.1 mM, and it can reduce the solubility of hydrophobic solutes until they precipitate. Compound-library studies measured water absorption rates for DMSO mixtures and how precipitated compounds could be redissolved.^[1]

Storage studies on small-molecule collections give useful reference points, although they did not test peptides. In one study of about 7,200 compounds stored as 20 mM DMSO solutions at room temperature, the probability of still detecting the compound by mass spectrometry was 92% after 3 months, 83% after 6 months and 52% after 1 year.^[2] A study of 10 mM stocks found that water was more important than oxygen in causing compound loss, found no significant loss after 11 freeze-thaw cycles when thawing was done under nitrogen with mixing and no difference between glass and polypropylene containers over 5 months.^[3]

Pure DMSO melts at about 18.5 °C, so stocks are solid in a refrigerator or freezer. A frozen stock is thawed completely and mixed before an aliquot is taken. Single-use aliquots in tightly closed tubes (see freeze-thaw cycles and aliquoting) limit both water uptake and repeated thawing.

Oxidation of thiols and methionine

DMSO is a mild oxidant. It is known to promote dimerization of thiols by oxidizing them to disulfides, and in a peptide reactivity assay the oxidation of a cysteine-derived reagent and a cysteine peptide increased as the DMSO concentration increased.^[4] A peptide with a free cysteine can therefore form disulfide-linked dimers in a DMSO stock; the guide on disulfide bonds and free thiols explains how free thiols are measured.

Methionine is also listed among the residues to watch in DMSO, because its oxidation to the sulfoxide adds 16 Da. Semax begins with methionine, and MOTS-c contains two, so an oxidized MOTS-c molecule would appear at about +16 or +32 Da in a mass spectrum. Mass spectrometry of a stored stock shows whether either change has occurred.

Final DMSO concentration and constant-vehicle dilution series

Because DMSO has a molar mass of 78.13 g/mol and a density of about 1.10 g/mL, neat DMSO is about 14 M. A final concentration of 0.1% (v/v) is therefore about 14 mM, and assay concentrations of 0.1 to 5% correspond to roughly 14 to 715 mM, making DMSO one of the main components of many assay buffers.^[5]

A worked example: a 10 mM stock diluted 1:1,000 gives 10 µM of peptide in 0.1% DMSO. For a concentration-response experiment, a common approach is to make the serial dilution in DMSO first (10, 3.33, 1.11, 0.370 and 0.123 mM for a 3-fold series) and then dilute each point 1:1,000 into buffer. Every well then contains 0.1% DMSO. If the series is instead made by diluting the top well 3-fold in buffer, the DMSO falls from 0.1% to 0.0012% across five points, an 81-fold change in solvent that runs in parallel with the peptide concentration.

A vehicle control with the same final DMSO percentage and no peptide measures any solvent effect. The dilution calculator handles the volume arithmetic, and the guide on Kd, Ki, IC50 and EC50 explains how concentration-response curves are fitted.

Precipitation on dilution

A peptide that dissolves well in DMSO can precipitate when the stock meets aqueous buffer. The concentration that stays in solution under these conditions is its kinetic solubility, which can be much lower than the stock concentration suggests. Low solubility in buffer and in DMSO has been linked to underestimated activity, variable data and disagreement between enzyme and cell assays, and the suggested strategies include better storage and handling of DMSO stocks and optimized dilution protocols.^[6]

Practical checks include adding the stock to buffer that is already being mixed, looking for turbidity, and centrifuging a test dilution and measuring the peptide left in the supernatant (see measuring peptide concentration). Hydrophobic peptides can also be lost to tube and plate surfaces once diluted; see peptide adsorption to labware.

Effects on proteins, cells and analytical methods

DMSO interacts with proteins. Measurements on four proteins showed preferential hydration at low DMSO concentrations, where the proteins remained native, and increasing DMSO binding to hydrophobic and aromatic side chains at higher concentrations, which correlated with unfolding, and the unfavorable interaction with native surfaces was linked to precipitation.^[7] In another study, low amounts of DMSO destabilized two proteins, changed the apparent binding properties of one, and influenced ionization in electrospray mass spectrometry.^[5]

Cells respond to DMSO as well. In five cell culture systems, DMSO above 1% reduced readouts in all cell types, while 0.25% and 0.5% produced inhibitory effects in some cell types and stimulatory effects in others; the authors stressed keeping solvent concentrations constant in serial dilutions.^[8]

DMSO also absorbs strongly in the far ultraviolet, so samples containing more than a trace of it are poorly suited to absorbance at 205 or 214 nm and to far-UV circular dichroism. A280 readings remain possible against a blank with the same DMSO content.

A record checklist

For results from a DMSO stock to be interpretable, record:

• Stock. Sequence, molecular weight and net peptide content used, mass weighed, DMSO grade and volume, and the calculated molar concentration.
• Storage. Temperature, container, aliquot size, number of thaw cycles and the age of the stock when used.
• Assay. Dilution scheme, final DMSO percentage in every sample, vehicle and untreated controls, and any visible precipitation.
• Checks. Mass spectrometry or concentration measurements on the stored stock, where available.

This guide describes general laboratory methods and published literature only. It is not a statement about the content or behaviour of any particular material. Terms used here are defined in the glossary, and questions can be sent to support.