Researcher preparing a microplate at a fluorescence imaging workstation

Longevity · Laboratory protocol

MOTS-c

A 16-residue mitochondrial-derived peptide encoded within 12S rRNA and translated in the cytoplasm. Published work links it to AMPK signalling and metabolic homeostasis.

For laboratory research use only. Material is supplied as a research reference standard and is not approved by Health Canada. Protocols below are compiled from published literature and supplier documentation; they are reference information, not medical advice. Anyone with a health condition or on medication should speak to a physician first.

Before you begin

• Lot-matched certificate and laboratory notebook
• Calibrated pipettes, low-binding tubes and suitable PPE
• Validated solvent, assay reagents and model-specific SOP
• Appropriate positive, negative and vehicle controls

Workflow

1Review the lot-matched COA and record identity, purity, batch number and received condition.
2Let the sealed vial equilibrate to room temperature before opening to limit condensation.
3Calculate the target stock concentration with C = mass / volume. Use only a solvent validated for the planned assay.
4Add solvent slowly down the vial wall, mix gently and avoid vortexing unless the validated method permits it.
5Prepare a log-spaced pilot concentration series; determine the usable range empirically instead of assuming a biological dose.
6Run the defined controls and technical replicates alongside every plate or experiment.
7Record preparation time, temperature, equipment, deviations and raw-data file locations.

Assay design

Bioenergetic endpoints such as oxygen-consumption rate or ATP production, normalized to protein content or viable cell number, with AMPK phosphorylation probed by immunoblot as the pathway readout.

Required controls

Vehicle-only negative control
Untreated baseline
Validated pathway control
Parallel viability measurement

Reported protocol

Standard: 5.0 mg once or twice per week
Timing: 1 to 2 hours prior to physical exercise
Route: Subcutaneous

Diluent

Sterile bacteriostatic water (0.9% benzyl alcohol) for multi-withdrawal stock, or sterile water for injection where a preservative-free stock is required by the assay.

Reconstitution concentrations

Solvent volume sets the stock concentration for this 40 mg vial. Lower volume gives a more concentrated stock and smaller working aliquots; higher volume gives a more dilute stock that is easier to measure accurately.

Stock concentration by solvent volume for a 40 mg vial
Solvent added	Stock concentration	Per 0.1 mL	Per unit (U-100)
1 mL	40 mg/mL	4 mg	400 mcg
2 mL	20 mg/mL	2 mg	200 mcg
3 mL	13.33 mg/mL	1.33 mg	133.33 mcg
5 mL	8 mg/mL	800 mcg	80 mcg

Working range

Cell work commonly spans the high-nanomolar to low-micromolar band, higher than receptor-agonist peptides because the proposed mechanism is not a cell-surface receptor.

Bioenergetic endpoints such as oxygen-consumption rate or ATP production, normalized to protein content or viable cell number, with AMPK phosphorylation probed by immunoblot as the pathway readout.

Stability

Lyophilized: Stable sealed and dry at the certificate temperature.
In solution: Establish empirically; aliquot to avoid freeze-thaw cycling.

Handling notes

Normalize every bioenergetic reading to protein or viable cell count. Raw OCR between wells is not comparable.
Seahorse-type assays need their own vehicle control on the same plate; edge wells drift.
Confirm which vial size you are working from before calculating; this material is supplied in more than one size.

Documentation checklist

Record lot and COA, mass balance, solvent and concentration, preparation timestamp, storage history, plate map, replicate plan, instrument settings, exclusions and deviations. Preserve raw data and analysis code with the experiment record.