Plasmid Map
Tools

Protein concentration calculator

A280 to mg/mL and µM, with ε from the sequence.

01

Your values

Paste the mature protein as measured, including any tag. FASTA headers, spaces and numbers are ignored.

Disulfides add 125 M⁻¹ cm⁻¹ per pair. Choose reduced for cytoplasmic proteins or samples with DTT or TCEP.

AU
cm

Micro-volume instruments usually report values normalized to 1 cm.

fold

1 if you measured the sample undiluted.

02

Example result

Protein concentration

0.18848mg/mL

13.168 µM of a 14,313 Da protein (129 residues: 6 Trp, 3 Tyr, 8 Cys).

Molar concentration
13.168 µM
ε₂₈₀ used
37,970 M⁻¹ cm⁻¹
ε₂₈₀, the other assumption
37,470 M⁻¹ cm⁻¹ (reduced)
Average mass
14313 g/mol
A280 of 1 mg/mL (0.1%)
2.6528
Calculation & assumptions

c = A₂₈₀ ÷ (ε × l) = 0.5 ÷ (37,970 M⁻¹ cm⁻¹ × 1 cm) = 13.168 µM; × 14313 g/mol = 0.18848 mg/mL

  • ε₂₈₀ = 5500 × Trp + 1490 × Tyr + 125 × cystines (Pace et al., Protein Science 1995), as ExPASy ProtParam and Biopython calculate it. Mass: average residue masses, as in Biopython. The sequence should be the mature protein as measured, including any tag.
  • A coefficient from the sequence is an estimate for an unfolded or typical folded protein; use a measured coefficient when you have one.
  • Measure against a blank of the same buffer. Readings between about 0.1 and 1.0 are the most reliable; dilute stronger samples and enter the dilution factor.
  • Nucleic acids, turbidity and some additives (for example Triton X-100) also absorb at 280 nm and raise the result. Check A260/A280 for nucleic acid contamination.

From an A280 reading to mg/mL

Proteins absorb ultraviolet light at 280 nm mainly through tryptophan and tyrosine, with a small contribution from disulfide bonds. The Beer–Lambert law turns a blank-corrected reading into a concentration: c = A₂₈₀ / (ε × l), where ε is the molar extinction coefficient in M⁻¹ cm⁻¹ and l is the path length in cm. Multiply by the molar mass to get mg/mL.

Paste the protein sequence and the calculator counts tryptophan, tyrosine and cysteine, estimates ε₂₈₀ with the method of Pace et al. (1995) and calculates the average mass, the same way ExPASy ProtParam and Biopython do. If your datasheet gives a coefficient, choose that instead: a molar ε, or the absorbance of a 1 mg/mL solution. For the same protein's isoelectric point, net charge and amino acid composition, use the protein calculator on Codon Chart.

Worked example: hen egg-white lysozyme

Mature lysozyme has 129 residues, including 6 tryptophans, 3 tyrosines and 8 cysteines that form 4 disulfide bonds. Its ε₂₈₀ is 6 × 5,500 + 3 × 1,490 + 4 × 125 = 37,970 M⁻¹ cm⁻¹, and its average mass is 14,313 g/mol.

A blank-corrected A₂₈₀ of 0.50 in a 1 cm cuvette gives 0.50 ÷ 37,970 = 13.2 µM. Multiplying by 14,313 g/mol gives 0.188 mg/mL. A 1 mg/mL lysozyme solution would read about 2.65 in 1 cm.

Disulfides, dilution and path length

Each cystine adds 125 M⁻¹ cm⁻¹. Choose All cysteines reduced for a protein without disulfides, such as most cytoplasmic proteins, or for samples in DTT or TCEP. For lysozyme the two assumptions differ by 1.3%.

If you diluted the sample before measuring, enter the fold dilution to get the stock concentration. A path shorter than 1 cm, such as a 0.2 cm cell, is corrected in the calculation. Micro-volume instruments usually report absorbance already normalized to 1 cm; check your instrument's setting.

When A280 is the wrong measurement

A protein with no tryptophan or tyrosine barely absorbs at 280 nm, so the calculator refuses it: use a colorimetric assay such as BCA or Bradford. Nucleic acids absorb strongly near 260 nm and also raise A280, so check the A260/A280 ratio. Detergents such as Triton X-100, some additives and turbidity add absorbance too. Readings between about 0.1 and 1.0 are the most reliable; dilute stronger samples and enter the dilution.

To make a working solution at a target concentration, use the dilution calculator. For small molecules, the molarity calculator converts between mass and molar concentration.

Sources: Pace CN, Vajdos F, Fee L, Grimsley G, Gray T. How to measure and predict the molar absorption coefficient of a protein. Protein Science 4, 2411–2423 (1995). Masses and coefficients match Biopython 1.85's ProtParam on four reference proteins.