Quick answer
How is MOTS-c reconstituted and stored? Store lyophilized MOTS-c sealed at −20 °C. To reconstitute, bring the vial to room temperature, add bacteriostatic water (for a multi-use vial) or sterile water (for immediate aliquoting) slowly down the glass, and swirl gently until clear. A 10 mg vial with 2 mL gives 5 mg/mL, about 2.3 mM. Keep bacteriostatic-water stock at 2–8 °C and use within about 28 days; for longer storage, freeze single-use aliquots at −20 °C or −80 °C and thaw each once.
MOTS-c is a short peptide with two methionines and a tryptophan in sixteen residues, which makes it more oxidation-sensitive than its size suggests. The practices below are designed to keep a MOTS-c stock chemically consistent from the first experiment to the last: choosing the right diluent, calculating concentration correctly, limiting freeze–thaw cycles and recording what was done. For laboratory research use only; not for human consumption.
What you are starting with: lyophilized MOTS-c
MOTS-c (mitochondrial open reading frame of the 12S rRNA type-c) is a 16-amino-acid peptide encoded within the mitochondrial 12S ribosomal RNA gene, first described by Lee and colleagues in 2015. Research-grade MOTS-c is made by solid-phase synthesis and supplied as a freeze-dried white powder under a crimped stopper. Structural detail is covered in MOTS-c chemical structure and synthesis.
| Property | Value | Why it matters for handling |
|---|---|---|
| Sequence | MRWQEMGYIFYPRKLR | Confirms identity against the COA |
| Length | 16 amino acids | Short; dissolves quickly |
| Molecular weight | ≈ 2,174.6 g/mol | Needed for molar conversions |
| Methionine residues | Two (positions 1 and 6) | Prone to oxidation to methionine sulfoxide |
| Tryptophan and tyrosine | One Trp, two Tyr | Light-sensitive aromatic side chains |
| Charge | Net positive (three Arg, one Lys) | Readily water-soluble; can adsorb to negatively charged glass |
| Cysteine | None | No disulfide chemistry to manage |
| Vial size | 10 mg | Basis for the tables below |
Before opening, match the lot number on the MOTS-c vial to the certificate in the COA library and note the reported purity and mass-spectrometry identity.
Solvent selection for MOTS-c reconstitution
Because MOTS-c carries a net positive charge and has no long hydrophobic stretch, it dissolves in plain water without difficulty. The choice of diluent is therefore about what happens after dissolution: how the stock will be stored and what the downstream assay tolerates.
| Diluent | When to use it | Advantages | Limitations |
|---|---|---|---|
| Bacteriostatic water (0.9% benzyl alcohol) | A stock vial that will be entered repeatedly over days to weeks | Preservative limits microbial growth between punctures | Benzyl alcohol must be accounted for in cell-based assays; include a vehicle control |
| Sterile water | Stock that will be aliquoted and frozen at once, or used the same day | No additives; cleanest for cell culture and analytical work | No preservative; not suitable as a refrigerated multi-use vial |
| Phosphate-buffered saline (PBS) | Final working dilutions | Physiological pH and ionic strength for assays | Not ideal for stock storage; phosphate buffers shift pH on freezing, and salts raise aggregation risk at high peptide concentration |
| DMSO, then aqueous dilution | Rarely needed for MOTS-c | Rescues peptides that will not dissolve in water | DMSO can oxidize methionine and should be avoided for methionine-containing peptides unless essential |
The last row deserves emphasis. DMSO is a common rescue solvent for hydrophobic peptides, but it is a mild oxidant toward thioethers. With two methionines in sixteen residues and good intrinsic water solubility, MOTS-c gains nothing from DMSO and risks something. If a batch genuinely resists dissolving, a few minutes of rest, gentle swirling or a small amount of dilute (0.1%) acetic acid is the better first step.
Step-by-step reconstitution protocol
Materials needed
- MOTS-c 10 mg vial and lot-matched COA
- Chosen diluent at room temperature
- Sterile syringe and needle or calibrated pipette with sterile tips
- 70% isopropyl alcohol swabs and gloves
- Sterile low-protein-binding microcentrifuge tubes for aliquots
- Optional: 0.22 µm low-protein-binding syringe filter
Step 1: Equilibrate the vial
Leave the sealed vial at room temperature for 15–30 minutes. This prevents condensation from forming inside when the stopper is pierced.
Step 2: Calculate your target volume
Decide the stock concentration first. Concentration (mg/mL) = 10 mg ÷ diluent volume (mL). The table gives common options, including the mass per graduation on a U-100 laboratory syringe scale (100 units = 1 mL).
| Diluent added | Concentration | Mass per 1 unit (0.01 mL) | Mass per 10 units (0.1 mL) | Units holding 1 mg |
|---|---|---|---|---|
| 1 mL | 10 mg/mL | 100 mcg | 1,000 mcg | 10 units |
| 2 mL | 5 mg/mL | 50 mcg | 500 mcg | 20 units |
| 2.5 mL | 4 mg/mL | 40 mcg | 400 mcg | 25 units |
| 4 mL | 2.5 mg/mL | 25 mcg | 250 mcg | 40 units |
| 5 mL | 2 mg/mL | 20 mcg | 200 mcg | 50 units |
| 10 mL | 1 mg/mL | 10 mcg | 100 mcg | 100 units |
The reconstitution calculator covers any other volume. A 2 mL fill (5 mg/mL) is a practical default: concentrated enough to limit adsorption losses, dilute enough to measure accurately.
Step 3: Add solvent carefully
Swab the stoppers and let them dry. Introduce the diluent slowly along the inside wall of the vial rather than directly onto the powder.
Step 4: Mix gently
Swirl or roll for 20–30 seconds and let the vial stand for a minute or two. Do not vortex or shake; agitation pulls air into the liquid and promotes methionine oxidation. The solution should be clear and colorless.
Step 5: Filter for cell-culture applications
If the stock was prepared in sterile water for cell work and sterility is critical, pass it through a 0.22 µm PES or PVDF filter. Expect to lose a small fraction of peptide to the membrane and the filter's hold-up volume, so filter the concentrated stock rather than dilute working solutions.
Step 6: Aliquot into single-use volumes
Dispense into low-bind tubes in volumes matched to one experiment, plus about 10% overage. For example, a 2 mL stock at 5 mg/mL can be split into twenty 100 µL aliquots of 0.5 mg each. Label each tube with compound, lot, concentration and date.
Step 7: Freeze promptly, or refrigerate a preserved vial
Move aliquots to −20 °C or −80 °C without delay. If you reconstituted with bacteriostatic water and intend to keep one multi-use vial, place it at 2–8 °C instead and skip the freezer.
Storage guidelines summary
| Storage condition | Duration | Notes |
|---|---|---|
| Lyophilized, −20 °C or colder | 12–24+ months | Sealed, dark, dry; preferred long-term condition |
| Lyophilized, 2–8 °C | Up to a few months | For vials that will be used soon |
| Lyophilized, ambient | Days (shipping) | Move to cold storage on receipt |
| Solution in bacteriostatic water, 2–8 °C | Up to about 28 days | Multi-use vial; keep boxed; shorter for sensitive assays |
| Solution in sterile water, 2–8 °C | Up to 24 hours | No preservative; aliquot and freeze instead |
| Aliquots, −20 °C | About 1–3 months | Single thaw |
| Aliquots, −80 °C | About 6 months | Single thaw; best option for long studies |
| Diluted working solution in buffer or medium | Same day | Prepare fresh; do not store |
The durations for solutions are conservative laboratory conventions, not validated shelf lives for a particular batch. The 28-day limit for bacteriostatic-water stocks reflects the standard for preserved multi-use containers under USP <797> and antimicrobial effectiveness testing under USP <51>. Where a protocol runs for months, plan around frozen aliquots or reconstitute a fresh vial partway through, and consider confirming integrity by HPLC.
Managing freeze–thaw cycles
Freezing is not a neutral event for a peptide in solution. As ice forms, solutes concentrate in the remaining liquid, pH can shift, and the peptide meets a large ice–water interface; on thawing, the process reverses. One cycle is generally tolerated. Several cycles add up to measurable loss through aggregation and adsorption.
- Size aliquots so that nothing needs to be refrozen.
- Thaw on ice or in the refrigerator, not in a warm water bath.
- Mix a thawed aliquot by gentle pipetting or inversion before use; solutes stratify during freezing.
- Avoid frost-free freezers for peptide storage if possible, since their defrost cycles warm the contents repeatedly.
- Keep a tally on the tube if a second thaw is ever unavoidable, and note it in the experimental record.
Concentration conversion reference
Cell-based studies of MOTS-c are usually reported in molar units. Convert with: molarity (µM) = [concentration (mg/mL) ÷ 2,174.6] × 1,000,000.
| Stock concentration | Molar equivalent | Stock volume for 1 mL of 10 µM working solution |
|---|---|---|
| 10 mg/mL | ≈ 4.60 mM | ≈ 2.2 µL |
| 5 mg/mL | ≈ 2.30 mM | ≈ 4.3 µL |
| 2 mg/mL | ≈ 920 µM | ≈ 10.9 µL |
| 1 mg/mL | ≈ 460 µM | ≈ 21.7 µL |
| 0.1 mg/mL | ≈ 46 µM | ≈ 217 µL |
The right-hand column uses C1V1 = C2V2. For concentrations in the nanomolar range, prepare an intermediate dilution rather than pipetting volumes below 1–2 µL. All values assume the weighed mass is 100% peptide. Lyophilized peptides contain counter-ions and residual moisture, so for precise molar work multiply by the net peptide content and purity stated on the COA.
Common mistakes to avoid
| Mistake | Consequence | Better practice |
|---|---|---|
| Dissolving in DMSO by default | Methionine oxidation | Use water; MOTS-c is water-soluble |
| Vortexing | Aeration, oxidation, aggregation | Swirl gently |
| Storing stock in PBS or medium | pH shifts on freezing; faster degradation; microbial growth | Store in water; dilute into buffer at time of use |
| Keeping a sterile-water solution in the fridge for weeks | Unpreserved solution can support microbial growth | Aliquot and freeze, or use bacteriostatic water |
| Repeated freeze–thaw of one tube | Cumulative loss of intact peptide | Single-use aliquots |
| Very dilute stocks in standard tubes | Cationic peptide adsorbs to surfaces | Keep stock ≥ 1 mg/mL; low-bind plastic |
| Clear vials left on the bench | Photo-oxidation of Trp and Met | Box or foil-wrap; minimize light |
| No label | Unknown concentration and age | Label at the time of reconstitution |
Related MOTS-c research
- MOTS-c research overview
- MOTS-c mechanism of action
- MOTS-c preclinical research
- MOTS-c research FAQ
- Where to buy MOTS-c
- Storing research peptides and how to mix peptides with bacteriostatic water
Summary
MOTS-c is easy to dissolve and easy to damage. Keep it lyophilized and frozen until needed, reconstitute in water without agitation, avoid DMSO, protect it from light and air, and either refrigerate a bacteriostatic-water vial for up to about four weeks or freeze single-use aliquots for longer work. Calculate concentrations from the vial mass and the volume actually added, convert to molar units with the molecular weight of 2,174.6 g/mol, and keep a record of each step.
References
- Lee C, Zeng J, Drew BG, et al. The mitochondrial-derived peptide MOTS-c promotes metabolic homeostasis and reduces obesity and insulin resistance. Cell Metab. 2015;21(3):443-454.
- Kim KH, Son JM, Benayoun BA, Lee C. The mitochondrial-encoded peptide MOTS-c translocates to the nucleus to regulate nuclear gene expression in response to metabolic stress. Cell Metab. 2018;28(3):516-524.
- United States Pharmacopeia. General Chapter <797> Pharmaceutical Compounding—Sterile Preparations. USP–NF.
- United States Pharmacopeia. General Chapter <51> Antimicrobial Effectiveness Testing. USP–NF.
- Manning MC, Chou DK, Murphy BM, Payne RW, Katayama DS. Stability of protein pharmaceuticals: an update. Pharm Res. 2010;27(4):544-575.
- Wang W. Instability, stabilization, and formulation of liquid protein pharmaceuticals. Int J Pharm. 1999;185(2):129-188.
- Goebel-Stengel M, Stengel A, Taché Y, Reeve JR Jr. The importance of using the optimal plasticware and glassware in studies involving peptides. Anal Biochem. 2011;414(1):38-46.
Frequently Asked Questions
How much bacteriostatic water should I add to a 10 mg MOTS-c vial?
Two milliliters is a practical default and gives 5 mg/mL, or about 2.3 mM. One milliliter gives 10 mg/mL and 5 mL gives 2 mg/mL. The volume only changes the concentration; the vial still contains 10 mg.
What is the best solvent for MOTS-c?
Water. Use bacteriostatic water for a multi-use refrigerated vial and sterile water when the stock will be aliquoted and frozen immediately or used in cell culture. MOTS-c is water-soluble, so organic solvents are rarely required.
Can MOTS-c be dissolved in DMSO?
It can, but it is not recommended. DMSO can oxidize methionine, and MOTS-c has two methionine residues. Because the peptide dissolves readily in water, DMSO adds risk without benefit.
How long does reconstituted MOTS-c last in the fridge?
In bacteriostatic water at 2–8 °C, the conventional working window is up to about 28 days. In unpreserved sterile water, use within 24 hours or freeze aliquots.
How should lyophilized MOTS-c be stored?
Sealed, in the dark, at −20 °C or colder for long-term storage. Refrigeration at 2–8 °C is acceptable for vials that will be reconstituted within a few months.
How many freeze–thaw cycles can MOTS-c tolerate?
Plan for one. Prepare single-use aliquots so that no tube is thawed twice. Each additional cycle increases the chance of aggregation, oxidation and adsorption losses.
How do I convert MOTS-c from mg/mL to µM?
Divide mg/mL by the molecular weight of 2,174.6 g/mol and multiply by 1,000,000. One mg/mL is roughly 460 µM, and 5 mg/mL is roughly 2.3 mM, before correcting for net peptide content.
Should MOTS-c solution be filtered?
Only when sterility of a sterile-water stock is critical, such as for cell culture. Use a 0.22 µm low-protein-binding filter on the concentrated stock and expect a small loss of material.
Why should I avoid vortexing MOTS-c?
Vortexing draws air into the solution and creates shear and interfaces that promote methionine oxidation and aggregation. Gentle swirling dissolves the peptide just as effectively.
What does properly reconstituted MOTS-c look like?
A clear, colorless solution with no visible particles. Cloudiness, a yellow tint or floating material indicates the solution should be discarded.
How many mcg of MOTS-c are in one unit on a U-100 syringe scale?
It depends on the concentration. At 5 mg/mL, one unit (0.01 mL) contains 50 mcg and ten units contain 500 mcg. At 2 mg/mL, one unit contains 20 mcg.