Researcher preparing a microplate at a fluorescence imaging workstation

Longevity · Laboratory protocol

NAD+

Nicotinamide adenine dinucleotide, a redox coenzyme and the substrate consumed by sirtuins, PARPs and CD38. Not a peptide.

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

NAD+/NADH ratio measurement, sirtuin or PARP activity assays, or bioenergetic endpoints. Because NAD+ is consumed by the enzymes being studied, substrate depletion over the incubation is a real confound and needs a timepoint series.

Required controls

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

Reported protocol

Concentration target: 100 mg/mL to 200 mg/mL
Standard: 50 mg to 100 mg daily, or 2 to 3 times per week
Loading: 50 mg daily for 10 consecutive days
Route: Subcutaneous or intramuscular

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.

Compound-specific: NAD+ solutions are pH-sensitive and degrade faster in alkaline conditions. Prepare fresh and check the pH of the final buffer rather than assuming it.

Reconstitution concentrations

Solvent volume sets the stock concentration for this 500 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 500 mg vial
Solvent added	Stock concentration	Per 0.1 mL	Per unit (U-100)
2 mL	250 mg/mL	25 mg	2500 mcg
3 mL	166.67 mg/mL	16.67 mg	1666.67 mcg
5 mL	100 mg/mL	10 mg	1000 mcg
10 mL	50 mg/mL	5 mg	500 mcg

Working range

Cell work runs the micromolar to millimolar band, orders of magnitude above the peptides in this catalog. Do not carry a peptide range across to this material.

Stability

Lyophilized: Hygroscopic. Keep tightly sealed and dry; reseal promptly after weighing.
In solution: Least stable material in this catalog in solution. Prepare fresh where the assay allows and do not hold dilute stock.

Handling notes

Prepare fresh. This is the one item here where an aged stock is the likely explanation for a failed run.
Check final buffer pH; alkaline conditions accelerate degradation.
Reseal the vial immediately. The powder takes up atmospheric moisture.
Include a timepoint series so substrate depletion is visible rather than silent.

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.