Peptide molarity and dilution: a worked lab example

Peptide molarity calculation takes three numbers: the mass of peptide you actually have, its molecular weight (MW), and the volume you dissolve it in. Molarity (mol/L) equals peptide mass (g) divided by MW (g/mol), divided by volume (L). From a stock solution you then reach working concentrations with C1V1 = C2V2. The step most people skip is correcting the weighed mass for net peptide content, because lyophilized powder is not 100% peptide. This guide works through every step for an in-vitro cell assay in µM units. You can check each number with our peptide calculator.

The formulas you need

A useful check: for any peptide, 1 µM equals MW ÷ 1000 µg/mL. For a 3,000 g/mol peptide, 1 µM is about 3 µg/mL.

Step 1: find the right molecular weight

Use the MW of the exact molecule in your vial. For this example we use thymosin alpha-1. FDA's 2024 briefing gives the free base as a 28-amino-acid, N-acetylated peptide, C₁₂₉H₂₁₅N₃₃O₅₅, MW 3108.3 g/mol.

Two traps:

• Free base vs salt. Peptides are often supplied as acetate or trifluoroacetate (TFA) salts. Check whether the MW on the certificate of analysis (CoA) is for the free peptide or the salt. Net peptide content already removes the counterion, so pair it with the free-base MW.
• Average vs monoisotopic mass. Mass-spec reports often show monoisotopic mass. For weighing, use the average MW.

Step 2: correct for net peptide content

Lyophilized peptide powder also contains water and counterions. Net peptide content is the fraction of the weighed mass that is actually peptide. HPLC purity is a different number: it tells you what share of the peptide material is the right sequence. Both are on a good CoA (see how to verify a peptide COA).

The counterion share can be large. In a 2018 Amino Acids study of antimicrobial peptides, the TFA salts contained about 156 to 318 µg of TFA per mg of sample, measured by ion chromatography. Ignore that, and your stock ends up weaker than its label.

Worked example. You have a vial containing 5.0 mg of thymosin alpha-1 powder. The CoA gives a net peptide content of 80% (an example figure; use your own lot's value).

• Peptide mass = 5.0 mg × 0.80 = 4.0 mg
• Moles = 4.0 mg ÷ 3108.3 g/mol = 0.001287 mmol = 1.287 µmol

Without the correction you would calculate 5.0 ÷ 3108.3 = 1.609 µmol. Every concentration downstream would then be overstated by 25%. Put another way, the real concentration would be only 80% of what the tube says.

Step 3: choose a stock concentration you can dissolve

A 1 mM stock of this peptide is 3.108 mg/mL. FDA's briefing reports a water solubility of 2 mg/mL for the free base, so 1 mM in water is above that figure. A 500 µM stock is a safer choice:

• Volume = 1.287 µmol ÷ 0.5 mM = 2.574 mL
• Mass concentration check: 4.0 mg ÷ 2.574 mL = 1.554 mg/mL, below 2 mg/mL

Solubility depends on the salt form, the pH and the solvent. Check the supplier's data or the literature for your exact material. If you need an organic co-solvent, record it, and include the same final solvent concentration in your vehicle control wells. Split the stock into single-use aliquots so you avoid repeated freeze-thaw cycles.

Step 4: dilute to a working concentration with C1V1 = C2V2

Say you need 2 mL of medium at 10 µM for a cell assay.

• C1 = 500 µM (stock), C2 = 10 µM, V2 = 2,000 µL
• V1 = (10 × 2,000) ÷ 500 = 40 µL of stock
• Add 40 µL of stock to 1,960 µL of medium. The total volume is V2, not V2 plus V1.

Use a two-step dilution when V1 becomes too small to pipette accurately. For 1 mL at 10 nM straight from the 500 µM stock, V1 would be 0.02 µL. Make an intermediate stock instead:

• Intermediate: 10 µL stock + 990 µL medium = 5 µM (a 1:100 dilution)
• Final: (0.010 µM × 1,000 µL) ÷ 5 µM = 2 µL intermediate + 998 µL medium = 10 nM

Step 5: build a serial dilution series

Concentration-response experiments usually use a fixed dilution factor. Here is a 10-fold series starting from the 10 µM working solution, plus a 2-fold plate series:

For the 2-fold column, transfer a volume into an equal volume of diluent (for example, 100 µL into 100 µL) instead of 100 into 900. The concentration in tube n is C1 ÷ factor⁽ⁿ⁻¹⁾. Mix well and change tips between steps, because any carryover error grows down the series.

Common mistakes

• Mixing units. mg ÷ g/mol gives mmol, not µmol. Write the units down at every step.
• Trusting the label mass. "5 mg" on a vial is usually gross powder weight, not peptide. Apply the net peptide content.
• Wrong MW. A salt-form MW combined with a net peptide correction double-counts the counterion.
• Adding V1 on top of V2. The final volume should be V2.
• No vehicle control. Any solvent or counterion in the stock also ends up in the wells.

Run your own numbers in the peptide calculator, which does the mg-to-mmol, molarity and C1V1 steps. For background on what peptides are and how they are made, see what are peptides, or browse the peptide library for MW data on each compound.

Frequently asked questions

Sources

1. FDA: Thymosin alpha-1 related bulk drug substances, PCAC briefing (4 December 2024): molecular formula, MW and solubility
2. Sikora K. et al. Counter-ion effect on antistaphylococcal activity and cytotoxicity of selected antimicrobial peptides. Amino Acids (2018)