What is Reconstitution?
Dissolving a lyophilised peptide in a measured volume of solvent to make a stock solution of known concentration.
By the APL Research Team · Updated
Reconstitution turns a lyophilised cake back into a solution, and in doing so it fixes the number every later calculation depends on: the stock concentration. Done carefully, the concentration is known to within the accuracy of the volume added and of the batch's net peptide content.
Choosing the solvent
The sequence decides the solvent. Short, charged peptides generally dissolve in water; acidic sequences may need a mildly basic buffer, basic ones a little dilute acetic acid, and very hydrophobic ones a small volume of an organic solvent such as DMSO before dilution. For aqueous stocks the usual choice is between sterile water, which adds nothing to the experiment and suits single-use stocks, and bacteriostatic water, whose benzyl alcohol allows repeated withdrawals but travels into every dilution. The solubility entry and the solubility troubleshooting guide cover sequences that resist.
The arithmetic
Concentration (mg/mL) = peptide mass (mg) ÷ volume (mL)
Working backwards from a molar target:
Volume (mL) = mass (mg) × 1,000 ÷ [MW (g/mol) × target concentration (mM)]
For a 5 mg vial of Semax (Met-Glu-His-Phe-Pro-Gly-Pro, 813.92 g/mol) and a 1 mM stock: 5 × 1,000 ÷ (813.92 × 1) = 6.14 mL. If only 80% of the powder were peptide, the vial would hold 4.0 mg and the same 1 mM target would need 4.91 mL.
| Target stock | Volume for 5 mg, all peptide | Volume for 5 mg at 80% net content |
|---|---|---|
| 5 mg/mL | 1.00 mL | 0.80 mL |
| 2 mg/mL | 2.50 mL | 2.00 mL |
| 1 mM (0.814 mg/mL) | 6.14 mL | 4.91 mL |
| 0.5 mM | 12.29 mL | 9.83 mL |
The bottom rows need more liquid than a small peptide vial holds. In that case the peptide is dissolved at a higher concentration first and diluted in a separate tube. The reconstitution calculator handles mass and volume combinations, and the molarity calculator the molar version.
Technique points that change the result
- Temperature. A vial taken from the freezer is left to reach room temperature before opening, so condensation does not wet the cake.
- Adding the diluent. It is run down the inside wall of the vial rather than jetted onto the cake, then the vial is swirled or rolled gently. Vigorous shaking foams the solution, and air–liquid interfaces promote aggregation.
- Waiting. A few minutes' contact time, then inspection against a dark background. Haze, flakes or a gel mean the peptide is not fully in solution, so the concentration is unknown.
- Recording. Concentration, solvent, date and batch go on the label. The solution is then kept at 2–8 °C or divided into single-use aliquots and frozen.
Common misunderstandings
- Clear is not the same as monomeric. Soluble oligomers are invisible to the eye.
- Diluent volume and final volume differ slightly, because the dissolved solid occupies some volume; for milligram fills the difference is small next to pipetting error.
- Reconstitution does not sterilise anything. The solution is only as clean as the diluent, the vial surface and the handling; the sterile technique guide covers the bench practice.
- The label mass may not be the peptide mass. Correcting for net peptide content is what makes a nominal 2 mg/mL stock a real one.
The peptide reconstitution guide gives the full step-by-step bench method.