How to reconstitute research peptides: the math
A lyophilized (freeze-dried) peptide arrives as a small dry cake or powder. Before it can be used in an assay it has to be dissolved in a measured volume of liquid to make a stock solution. The amount of liquid you add decides the concentration, and every later pipetting step depends on getting that number right.
The one formula
Everything on this page comes from a single division:
Two shortcuts make the rest easy:
- 1 mg/mL is the same as 1 µg/µL. So a 5 mg/mL stock holds 5 µg of peptide in every µL.
- Volume needed = amount wanted ÷ concentration. Keep both in the same units (µg and µg/µL, or mg and mg/mL).
Concentration tables for 5, 10, 20 and 30 mg vials
The stock concentration you get from each common vial size, by the volume of liquid added:
| Vial | 1 mL added | 2 mL added | 3 mL added | 5 mL added |
|---|---|---|---|---|
| 5 mg | 5 mg/mL | 2.5 mg/mL | 1.67 mg/mL | 1 mg/mL |
| 10 mg | 10 mg/mL | 5 mg/mL | 3.33 mg/mL | 2 mg/mL |
| 20 mg | 20 mg/mL | 10 mg/mL | 6.67 mg/mL | 4 mg/mL |
| 30 mg | 30 mg/mL | 15 mg/mL | 10 mg/mL | 6 mg/mL |
And the volume of that stock that contains 100 µg of peptide (a common reference amount for planning assays):
| Vial | 1 mL added | 2 mL added | 3 mL added | 5 mL added |
|---|---|---|---|---|
| 5 mg | 20 µL | 40 µL | 60 µL | 100 µL |
| 10 mg | 10 µL | 20 µL | 30 µL | 50 µL |
| 20 mg | 5 µL | 10 µL | 15 µL | 25 µL |
| 30 mg | 3.33 µL | 6.67 µL | 10 µL | 16.67 µL |
Values are rounded to two decimal places. They assume the vial holds exactly its stated amount of peptide; see the next section for why that is rarely the whole story.
A worked example
An experiment calls for 250 µg of peptide per well. You have a 10 mg vial and add 2 mL of liquid.
- Concentration: 10 mg ÷ 2 mL = 5 mg/mL, which is 5 µg/µL.
- Volume per well: 250 µg ÷ 5 µg/µL = 50 µL.
- Wells per vial: 10,000 µg ÷ 250 µg = 40, before allowing for losses in pipette tips and the vial wall.
If 50 µL is awkward to pipette accurately, change the volume you add at the start rather than rounding later. Adding 4 mL instead gives 2.5 µg/µL and 100 µL per well.
Our peptide calculator runs the same arithmetic if you prefer not to work it by hand.
Why "10 mg" may not mean 10 mg of peptide
The number on a vial is normally the gross weight of the powder. Freeze-dried peptide also contains bound water and counter-ions (usually trifluoroacetate or acetate left from purification), which the scale weighs but which are not peptide. The share that is real peptide is the net peptide content, and it is often well below 100% even when the peptide itself is very pure.
When your work needs an exact concentration, correct for it:
For example, a 10 mg vial with 80% net peptide content holds about 8 mg of peptide, so 2 mL of liquid gives 4 mg/mL, not 5. If the certificate reports net peptide content, use it; if it does not, note that your concentrations are based on gross weight. Our guide to reading a certificate of analysis explains where to find this figure.
Choosing the liquid
- Sterile water suits many short, water-soluble peptides when the whole stock will be used or frozen in portions straight away.
- Bacteriostatic water is sterile water with 0.9% benzyl alcohol, which slows microbial growth in a vial that will be opened more than once. Check that benzyl alcohol will not interfere with your assay.
- Hydrophobic or highly charged sequences may need a dilute acid, a dilute base or a small amount of an organic co-solvent such as DMSO to dissolve fully. Solubility depends on the sequence, so check the data for the specific peptide first.
Technique that protects the math
- Let a cold vial reach room temperature before opening it, so moisture does not condense on the powder.
- Measure the liquid with a calibrated pipette rather than estimating from a graduated tube.
- Run the liquid slowly down the inside wall of the vial instead of straight onto the powder.
- Swirl or roll gently until the solution is clear. Hard shaking can cause foaming and aggregation.
- Write the concentration, the date and the lot number on the vial before it goes into storage.
Common mistakes
- Mixing up mg and µg. They differ by a factor of 1,000. Convert everything to one unit before dividing.
- Ignoring net peptide content when comparing results between lots or suppliers.
- Rounding early. Keep full figures until the final step.
- Losing track of which vial is which. An unmarked vial of clear liquid can be anything.
Once your stock is made, how you store it matters as much as how you made it. See storing peptides: powder vs mixed.