The one formula that matters
Lyophilized peptides arrive as a dry powder with a stated mass: 5 mg, 10 mg, and so on. Reconstitution is nothing more than dissolving that mass in a known volume of bacteriostatic water so that every unit of volume contains a known quantity of peptide:
Concentration (mg/mL) = peptide mass (mg) ÷ diluent volume (mL)
Everything else is rearranging that equation. Add 2 mL of BAC water to a 5 mg vial and the resulting solution is 2.5 mg/mL. Add 1 mL instead and it is 5 mg/mL. Neither is more “correct”: the choice only changes how much volume corresponds to a given quantity in later work.
Worked example
Suppose a protocol calls for aliquots containing 250 mcg (0.25 mg) of peptide from a 5 mg vial.
- Choose a convenient concentration. With 2 mL of diluent: 5 mg ÷ 2 mL = 2.5 mg/mL.
- Convert the target quantity: 0.25 mg ÷ 2.5 mg/mL = 0.1 mL (100 µL) per aliquot.
- Sanity-check the total: 2 mL of solution ÷ 0.1 mL = 20 aliquots of 250 mcg, which accounts for the full 5 mg. If the numbers don’t reconcile, a conversion slipped somewhere.
Quick reference
For a 5 mg vial:
| BAC water added | Resulting concentration | Volume containing 100 mcg |
|---|---|---|
| 1 mL | 5 mg/mL | 20 µL |
| 2 mL | 2.5 mg/mL | 40 µL |
| 3 mL | 1.67 mg/mL | 60 µL |
| 5 mL | 1 mg/mL | 100 µL |
Doubling the vial mass halves the volume per unit quantity at the same diluent volume; scale accordingly.
Technique notes
- Run the water down the vial wall, not directly onto the lyophilized cake. A direct jet can shear delicate peptide chains.
- Swirl, never shake. Gentle rotation dissolves the cake in a minute or two. Vigorous agitation can cause foaming and degradation.
- Bacteriostatic vs. sterile water: BAC water contains 0.9% benzyl alcohol, which suppresses microbial growth in a multi-use vial. Plain sterile water has no preservative and suits single-use preparation only.
- Label immediately with compound, concentration, and reconstitution date. An unlabeled vial in a shared freezer is a discarded vial.
Common errors
The two mistakes we see most often are unit slips (mg where mcg was intended, a thousand-fold error) and dividing in the wrong direction (volume by mass instead of mass by volume). Both are caught by the reconciliation step in the worked example above: total volume divided by aliquot volume should always equal total mass divided by aliquot mass.
Wednesday supplies bacteriostatic water (30 mL) and research-grade lyophilized peptides with third-party COAs. Research use only.