Laboratory basics
Reconstitution, explained
How lyophilised (freeze-dried) peptides are brought into solution for in vitro work, and the arithmetic behind concentration.
Most research peptides are supplied as a lyophilised powder: the peptide is freeze-dried so it stays stable in storage and transport. Before it can be used in an assay it has to be dissolved in a liquid, a step called reconstitution. This page covers the laboratory principles and the arithmetic. It is not instruction for human or animal use.
The diluent
The liquid used to dissolve the peptide is called the diluent. Which one a laboratory uses depends on the peptide's solubility and on how long the solution has to last.
| Diluent | What it is | Typical laboratory use |
|---|---|---|
| Sterile water | Water with no additives | Single-use solutions |
| Bacteriostatic water | Sterile water with 0.9% benzyl alcohol, which inhibits bacterial growth | Multi-use vials kept refrigerated |
| Dilute acetic acid or other buffers | Mildly acidic or buffered solutions | Peptides that do not dissolve well in water |
The supplier's Certificate of Analysis and solubility notes are the reference for a specific batch.
Principles of handling
- Equilibrate. Let a refrigerated vial reach room temperature before opening to limit condensation.
- Work aseptically. Clean the stopper and work surface; use sterile transfer equipment.
- Add the diluent slowly down the inside wall of the vial, rather than directly onto the powder.
- Swirl gently, do not shake. Vigorous agitation can denature some peptides or cause foaming.
- Inspect. A properly dissolved solution is clear. Cloudiness or particles suggest incomplete dissolution or a problem with the material.
- Label and store. Record the concentration and date. Lyophilised peptides are generally stored frozen or refrigerated and protected from light; reconstituted solutions are generally refrigerated and have a shorter usable life. Follow the batch documentation.
The arithmetic
Concentration is mass divided by volume:
concentration (mg/mL) = peptide mass (mg) ÷ diluent volume (mL)
A 5 mg vial dissolved in 2 mL gives 2.5 mg/mL, which is 2,500 µg/mL. To find the volume that contains a given mass, divide the mass by the concentration: 250 µg ÷ 2,500 µg/mL = 0.1 mL (100 µL).
Concentration and volume calculator
Laboratory dilution arithmetic (C = m / V). Results are in mg/mL, mL and µL only.
Why accuracy matters
Small errors compound. Reading 2 mL as 1 mL doubles the concentration of every aliquot drawn from that vial. Laboratories record the lot number, the exact volume added and the date on every reconstituted vial so that results can be traced and repeated.