Start with the COA, not the vial label
A vial labeled 5 mg contains 5 mg of lyophilized powder — not necessarily 5 mg of peptide. Residual water and counter-ions left from purification occupy part of that mass. The figure to calculate from is net peptide content, reported separately on the Certificate of Analysis.
A vial reported at 5 mg gross with 87% net peptide content holds roughly 4.35 mg of peptide. Ignoring that difference introduces a systematic ~13% concentration error into every downstream calculation — an error that looks like biological variability and is not.
Choosing a solvent
Solubility is a property of the sequence, not of peptides in general. Charge distribution and hydrophobicity determine what will dissolve the compound cleanly, and the wrong solvent produces cloudiness, particulate, or silent partial dissolution.
| Solvent | Typical use | Notes |
|---|---|---|
| Sterile water | Neutral, well-behaved sequences | No preservative — prepare single-use aliquots |
| Bacteriostatic water | Multi-draw storage of stable sequences | 0.9% benzyl alcohol limits microbial growth; confirm sequence compatibility |
| Dilute acetic acid | Basic or poorly soluble sequences | 1–10 mM lowers pH to assist dissolution |
| Ammonium bicarbonate | Acidic sequences | Raises pH for peptides with low isoelectric points |
| DMSO | Highly hydrophobic sequences | Effective but assay-limiting above low % v/v; dissolve, then dilute |
| PBS or assay buffer | Assay-matched preparation | Buffer salts promote aggregation in some sequences |
The general principle is to move pH away from the peptide’s isoelectric point, where solubility is at its minimum: acidify basic sequences, alkalinize acidic ones. Begin with the least aggressive solvent that works and escalate only if the cake will not dissolve.
Calculating concentration
Concentration in mg/mL is net peptide mass divided by solvent volume. Using the worked example above, 4.35 mg of peptide in 2 mL of solvent yields 2.175 mg/mL, or 2,175 µg/mL.
Record the volume actually added rather than the volume intended — pipetting into a small vial with a partial vacuum rarely delivers exactly the nominal amount. Note the solvent, the lot number, and the date alongside the concentration; a stock solution with no provenance is unusable in a reproducible workflow.
Technique
Add solvent slowly down the inner wall of the vial rather than directly onto the cake, and let the material dissolve without agitation. Ten to twenty minutes at room temperature is typical for a well-matched solvent.
Do not shake or vortex aggressively. Mechanical shear and repeated exposure at the air–liquid interface both drive aggregation and oxidation. Gentle rolling or swirling is sufficient; a brief low-speed spin collects material adhering to the stopper.
When a peptide will not dissolve
Visible cloudiness or particulate after 30 minutes indicates solvent mismatch, not insufficient time. Adjust pH in the appropriate direction, or use brief sonication in a cool water bath.
Do not apply heat. Warming to force dissolution accelerates the same hydrolysis and deamidation reactions that lyophilization exists to prevent, and a clear solution obtained by heating may contain substantially less intact peptide than the cake did.
After reconstitution
A reconstituted peptide is markedly less stable than the lyophilized powder — water is the reactant in most degradation routes. Aliquot immediately into single-use volumes, label each with compound, concentration, solvent, and date, and freeze.
Repeated freeze–thaw of a single stock is one of the most common causes of unexplained potency loss between experiments. Aliquoting at the moment of reconstitution is the cheapest insurance available. See peptide storage and stability for temperature and shelf-life detail.
Net peptide content, on every lot
Every Ethos Bio compound ships with an independent Certificate of Analysis reporting net peptide content — the figure your concentration math should start from.
Frequently asked questions
What is the best solvent for reconstituting a research peptide?
Sterile water suits most well-behaved sequences. Poorly soluble sequences may require dilute acetic acid (basic peptides), dilute ammonium bicarbonate (acidic peptides), or a small percentage of DMSO for highly hydrophobic sequences. Solubility is sequence-specific, so consult the product documentation and Certificate of Analysis before choosing. For laboratory research use only.
How do you calculate peptide concentration after reconstitution?
Divide net peptide mass by solvent volume, using the net peptide content from the Certificate of Analysis rather than the vial’s gross label weight. A 5 mg vial at 87% net peptide content contains about 4.35 mg of peptide, so 2 mL of solvent yields roughly 2.18 mg/mL.
Why should reconstituted peptides be aliquoted?
Reconstituted peptides are far less stable than lyophilized powder, and each freeze–thaw cycle degrades material further. Splitting the stock into single-use aliquots at the time of reconstitution means each aliquot is thawed only once.