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Peptide Concentration and Stock Solution Calculator

Written and reviewed by LifeTein’s peptide chemistry team, PhD-level scientists working in our Somerset, New Jersey laboratory · Last reviewed

Two calculations that decide whether your concentration is right, and that the peptide calculator does not cover: working out a concentration from an A280 reading, and working out how much solvent to add to a vial once you account for the fact that not all of the powder is peptide.

1. Sequence → extinction coefficient

Absorbance at 280 nm comes almost entirely from tryptophan and tyrosine, with a small contribution from disulfides. Paste a sequence to get the coefficient the next calculator needs.

Spaces and line breaks are ignored.

2. A280 → concentration

Read the absorbance against the same buffer as a blank. Dilute until the reading falls below about 1.0, and enter the dilution factor.

Override for modified peptides.

3. Vial → stock solution

A lyophilized vial contains peptide plus counter-ions and water. Net peptide content is the fraction that is actually peptide; it is on your certificate of analysis when it has been measured.

Typical range 50–90% for a TFA salt.

If you do not know the net peptide content

The default of 80% above is only a placeholder. Real values for a TFA salt commonly fall between 50% and 90%, so assuming the wrong one can put your concentration out by a factor of nearly two. Where the number matters, have it measured — see what is net peptide content and quality and documentation.

What these calculations assume

  • A280 needs an aromatic residue. With no tryptophan or tyrosine the peptide does not absorb usefully at 280 nm and the method simply does not apply. The first calculator says so when that happens.
  • Tyrosine-only sequences are less reliable. The coefficient is smaller and the relative error larger, so treat the result as approximate.
  • The molecular weight must match the peptide you actually have. The sequence box gives the unmodified free-acid value. Amidation, acetylation, a label or a disulfide all shift it, so take the figure from your certificate of analysis or from the peptide calculator for modified peptides.
  • The peptide has to be fully dissolved. Undissolved material reads low and makes every number here wrong. See how to dissolve peptides.
  • Scattering inflates A280. An aggregated or cloudy sample reads high. Centrifuge and look at the pellet before trusting a reading; see stability and degradation.

Related pages

References

  1. Pace, C. N., Vajdos, F., Fee, L., Grimsley, G. & Gray, T. How to measure and predict the molar absorption coefficient of a protein. Protein Sci. 4, 2411-2423 (1995).
  2. Gill, S. C. & von Hippel, P. H. Calculation of protein extinction coefficients from amino acid sequence data. Anal. Biochem. 182, 319-326 (1989).

Frequently asked questions

How do I work out peptide concentration from A280?

Divide the absorbance by the extinction coefficient and the path length. The coefficient comes from the sequence: 5500 per tryptophan, 1490 per tyrosine and 125 per disulfide. A peptide with neither tryptophan nor tyrosine cannot be measured this way.

Why is my peptide concentration lower than the vial weight suggests?

Because a lyophilized vial contains counter-ions and water as well as peptide. Net peptide content for a TFA salt commonly falls between 50 and 90 percent, so using the gross weight overestimates the amount of peptide.

What extinction coefficient should I use for a peptide with no Trp or Tyr?

None. Such a peptide does not absorb usefully at 280 nm. Use amino acid analysis or a colorimetric assay instead.

Does a disulfide change the extinction coefficient?

Yes, slightly. Each cystine contributes about 125 to the coefficient. Free cysteines contribute essentially nothing.

Research use only. LifeTein peptides and proteins are supplied for laboratory research use. They are not for human or veterinary use, and not for diagnostic or therapeutic use. See our research use policy.