Stock solutions and serial dilutions

Almost no experiment uses a peptide at the concentration it was dissolved at. A concentrated stock is made once, from a weighed or whole-vial amount, and diluted to working concentrations as needed. Doing that well is mostly arithmetic and pipette discipline, and the errors it introduces are systematic rather than random: a stock that is 10% strong makes every point on a curve 10% strong. This article covers the stock, the dilution equation, serial dilutions and their compounding error, and the practical habits that keep the numbers honest.

Why make a stock at all

Weighing is most accurate when the mass is large relative to the balance's resolution, and dissolving a whole vial removes weighing altogether. So the accurate step is to make a concentrated solution, typically 1–10 mM for a small peptide, and the convenient step is to dilute it. A stock also freezes well in aliquots, while a working solution at micromolar concentration loses peptide to the walls of its tube within hours; storing peptide solutions gives the figures.

The dilution equation

Concentration times volume is conserved: C₁V₁ = C₂V₂. To make volume V₂ at concentration C₂ from a stock at C₁, take V₁ = C₂V₂ ÷ C₁ of stock and make up to V₂. The units of concentration must match on both sides, which is where the unit ladder matters: a 1 mM stock is 1,000 µM, so 10 µM in 1 mL needs 10 µL of stock and 990 µL of diluent.

Volumes of a 1 mM stock to make 1 mL at a working concentration

Working concentration	Dilution factor	Stock to take	Diluent	Comment
100 µM	10	100 µL	900 µL	Comfortable for any pipette
10 µM	100	10 µL	990 µL	A 10 µL pipette's range; still accurate
1 µM	1,000	1 µL	999 µL	At the limit: make it in two steps instead
100 nM	10,000	0.1 µL	—	Not pipettable; serial dilution required

Serial dilutions

A serial dilution makes a large factor as a chain of small ones: 1:10, then 1:10 again, and so on. Each step uses pipettable volumes, and the concentrations fall in a regular series that suits a concentration–response experiment in vitro. Two rules keep it accurate. Mix each tube fully before taking from it; an unmixed tube propagates its gradient into every tube below. And never dilute by more than about 1:10 in a single step from small volumes, because the pipetting error on the small volume dominates.

The error compounds. If each step is accurate to 2%, six steps are accurate to about 1.02⁶, which is 12.6% at worst if the errors run the same way, or about 5% (2% × √6) if they are random. For a series that spans six orders of magnitude, measuring the stock's concentration by absorbance and preparing the low end from a fresh intermediate are the two practical defenses.

Pipettes, and the volumes they are good at

An air-displacement pipette is most accurate in the upper part of its range and least at the bottom: a 2–20 µL pipette at 2 µL may be off by 3–5%, at 20 µL by under 1%. Choosing the pipette whose range the volume falls high in, pre-wetting the tip, and pipetting viscous solvents such as DMSO slowly or with reverse pipetting all reduce the error. ISO 8655 sets the tolerances manufacturers calibrate to, and a laboratory that checks its pipettes gravimetrically every few months knows which ones to trust at the low end.

Intermediate stocks

A tidy scheme for a wide range: a primary stock at 1 mM, an intermediate at 10 µM made in one 1:100 step from 10 µL plus 990 µL, and working solutions from the intermediate. Each step stays within pipette range, the intermediate can be made fresh each day from a frozen aliquot of the primary stock, and the low concentrations never sit in a tube long enough to adsorb.

What to dilute into

The diluent sets the final solvent composition. A peptide dissolved in DMSO at 10 mM and diluted 1:1,000 into an aqueous buffer gives 0.1% DMSO in the final solution, usually tolerable in cell-based work; diluting 1:10 gives 10%, which is not. If a peptide was dissolved with a little acid or base, the diluent's buffer must be strong enough to absorb it; a 10 mM phosphate buffer absorbs 1% of a 0.1 M acetic acid stock with a pH shift of a few hundredths. The buffers article covers buffer capacity; which solvent for which peptide covers the organic co-solvents.

Recording it

A dilution scheme written as a table, with the stock's lot number and its measured or calculated concentration, is the record that lets a result be reproduced. The lot number ties the stock to a certificate; the certificate's net peptide content ties the weighed mass to the peptide in it. Without the lot number, the chain back to the material is broken at the first link.

Frequently asked questions

How concentrated should a stock be?

As concentrated as the peptide will stay dissolved and stable, usually 1–10 mM for a short peptide and 0.1–1 mM for a large or hydrophobic one. A stock ten to a hundred times the highest working concentration leaves room to dilute in one or two steps.

Can I pipette 1 µL of stock?

You can, with a pipette made for it, but the error is several percent. Two steps of 1:30, or a 1:100 intermediate, are more accurate.

Does a serial dilution lose peptide to the tubes?

At low concentration, yes, and the loss is a larger fraction of less material. Low-binding tubes, a carrier protein in the diluent where the assay allows it, and making the low end fresh all limit it.

Why do my replicate curves shift together?

A shift shared by every point points at the stock: its concentration, its net peptide content correction, or an unmixed first tube. Random scatter points at the pipetting.

Sources & Further Reading

1. International Organization for Standardization. ISO 8655-2:2022, Piston-operated volumetric apparatus, Part 2: Pipettes; and ISO 8655-6:2022, Gravimetric reference measurement procedure.
2. Goebel-Stengel M, Stengel A, Taché Y, Reeve JR. The importance of using the optimal plasticware and glassware in studies involving peptides. Analytical Biochemistry 2011;414(1):38–46. doi:10.1016/j.ab.2011.02.009
3. Tjernberg A, Markova N, Griffiths WJ, Hallén D. DMSO-related effects in protein characterization. Journal of Biomolecular Screening 2006;11(2):131–137. doi:10.1177/1087057105284218
4. International Union of Pure and Applied Chemistry. Amount concentration. IUPAC Compendium of Chemical Terminology (the "Gold Book"). doi:10.1351/goldbook.A00295

Cosmic Peptides. (2026, October 30). Stock solutions and serial dilutions. Cosmic Peptides Research Library. Retrieved October 30, 2026, from https://cosmicpeptides.com/research/methods/serial-dilutions-and-stock-solutions

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  author       = {{Cosmic Peptides}},
  title        = {Stock solutions and serial dilutions},
  howpublished = {Cosmic Peptides Research Library},
  year         = {2026},
  month        = oct,
  url          = {https://cosmicpeptides.com/research/methods/serial-dilutions-and-stock-solutions},
  urldate      = {2026-10-30},
  note         = {Last reviewed 2026-10-30}
}

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