Most reproducibility failures are handling failures, not material failures. This bench guide covers diluent choice, concentration maths, storage temperature and freeze-thaw discipline.
A 99% pure peptide handled badly produces the same unusable dataset as an 80% pure peptide handled well. Handling protocol is part of the experiment, not preparation for it.
The concentration calculation
Concentration equals net peptide mass divided by diluent volume. Use the net mass on the certificate — the figure that accounts for counterion and water content — not the nominal label mass. A 5 mg vial reconstituted with 2 mL gives 2.5 mg/mL.
Choosing a diluent
| Diluent | Typical use | Working window |
|---|---|---|
| Bacteriostatic water | Multi-draw research handling | Extended, benzyl-alcohol preserved |
| Sterile water | Single-session use | Short — no preservative |
| Acetic acid solution | Poorly soluble hydrophobic sequences | Sequence dependent |
Storage discipline
- Keep lyophilised powder at -20°C, sealed and light-protected until the day of use.
- Add diluent down the vial wall — never inject directly onto the powder cake.
- Swirl to dissolve; do not shake, which shears longer sequences.
- Refrigerate reconstituted solution at 2-8°C and log the reconstitution date.
- Aliquot before freezing so no vial sees more than one freeze-thaw cycle.
Handling FAQ
How many freeze-thaw cycles are acceptable?
Plan for one. Each cycle risks aggregation and degradation, so aliquoting at first reconstitution is the standard control.
Why swirl rather than shake?
Shaking introduces shear and foaming that can denature or aggregate longer peptide sequences. Gentle swirling dissolves without that risk.
