Extinction coefficient calculator

How to calculate protein concentration from A280

Paste the protein sequence, enter the absorbance your spectrophotometer read at 280 nm, and set the path length and any dilution. The calculator predicts the molar extinction coefficient from the sequence, then applies the Beer-Lambert law and returns the concentration in M, µM and mg/mL, with reduced and oxidized cysteine assumptions side by side.

Hen egg-white lysozyme makes a good first check because both its sequence and its absorbance are well known. Paste the 129-residue mature chain (UniProt P00698 without its signal peptide):

>lysozyme
KVFGRCELAAAMKRHGLDNYRGYSLGNWVCAAKFESNFNTQATNRNTDGSTDYGILQINSRWWCNDGRTPGSRNLCNIPCSALLSSDITASVNCAKKIVSDGNGMNAWVAWRNRCKGTDVQAWIRGCRL

Set A280 to 0.65 and leave Path length (cm) at 1, Dilution factor at 1 and Concentration basis at Oxidized coefficient. The result row reads:

All four lysozyme disulfides are formed in the folded protein, so the oxidized coefficient is the right one here. The reduced columns, 17.347 µM and 0.248 mg/mL, show how little the cystine term matters for a protein with six tryptophans: about 1%.

The Beer-Lambert calculation

Absorbance is proportional to concentration and path length:

Rearranged for concentration, with the dilution factor $d$ applied to get back to the undiluted sample:

For lysozyme, $0.65 / (37{,}970 \times 1) = 1.712 \times 10^{-5}$ M, or 17.12 µM. Multiplying molar concentration by molecular weight in g/mol gives g/L, which is the same number as mg/mL: $1.712 \times 10^{-5} \times 14{,}313 = 0.245$ mg/mL.

Dilution matters most for concentrated stocks, which saturate the detector. Suppose an antibody stock is diluted 1:10 (10 µL into 90 µL of buffer, so Dilution factor is 10) and the diluted sample reads 0.742 in a 1 cm cuvette. With the trastuzumab sequence from the antibody section below, the calculator returns 34.451 µM and 5.000 mg/mL for the stock.

Microvolume spectrophotometers such as the NanoDrop usually report absorbance already normalized to a 1 cm path. Leave Path length (cm) at 1 for those readings and change it only when you enter a raw value from a shorter or longer cuvette.

What is an extinction coefficient?

A 1 mg/mL solution of bovine serum albumin absorbs about 0.65 at 280 nm. The same mass of lysozyme absorbs 2.65. Lysozyme is not four times more concentrated; it carries six tryptophans in 129 residues, while BSA carries two in 583. The extinction coefficient is the number that captures this difference, and it is what turns an absorbance reading into a concentration.

The molar extinction coefficient ($\varepsilon$), also called the molar absorption coefficient or molar absorptivity, is the absorbance of a 1 M solution in a 1 cm path at a given wavelength. Its units follow from the Beer-Lambert law, because absorbance itself has no units:

Molar values suit binding and kinetics work, where you need µM. Mass values suit formulation and protein yield, where you need mg/mL. The two are linked by molecular weight.

How the extinction coefficient is predicted from sequence

At 280 nm, nearly all protein absorbance comes from three chromophores: the indole ring of tryptophan, the phenol ring of tyrosine and the disulfide bond of cystine. Phenylalanine absorbs too weakly at this wavelength to count. The predicted coefficient is a weighted sum of residue counts:

These are the values Pace and colleagues fitted to 116 measured coefficients of 80 folded proteins in water, and they are the values this calculator uses, as does ExPASy ProtParam. They describe each chromophore as it sits on average inside a folded protein, not as a free amino acid.

Gill and von Hippel took the other route in 1989. They predicted the coefficient of the unfolded protein in 6 M guanidine hydrochloride from model-compound values measured by Edelhoch, then corrected it to the native state with a measured absorbance ratio. Calibrated against 18 globular proteins, their method was accurate to about ±5% in most cases.

The two columns differ because a residue's absorbance depends on its surroundings. Buried tryptophans absorb less than tryptophan in denaturant, and tyrosines absorb more. Pace's equation folds that average shift into the coefficients, so it predicts the native protein directly; the Edelhoch values need the unfolded protein to be measured as well.

Reduced and oxidized values

A free cysteine thiol absorbs essentially nothing at 280 nm. A disulfide bond adds 125 $\text{M}^{-1}\,\text{cm}^{-1}$, so cystine is counted per bond, never per cysteine. The calculator therefore reports two coefficients for every sequence:

• Reduced: every cysteine is a free thiol and the cystine term is zero. Use it for cytoplasmic proteins and for samples in DTT, TCEP or another reducing agent.
• Oxidized: every possible pair of cysteines forms a disulfide, which is floor(Cys / 2) bonds. Use it for secreted proteins, antibodies and other proteins with native disulfides.

BSA shows why the distinction can matter. Its mature chain has 35 cysteines, and the oxidized value assumes 17 disulfides with one cysteine left over, which happens to match the real protein: 17 disulfides and a free Cys34. The coefficient rises from 40,800 to 42,925, a 5% change in every concentration you calculate. When you know a protein forms fewer bonds than the maximum, choose Specified disulfide count and enter the number.

E 0.1%: the mg/mL form

Dividing the molar coefficient by molecular weight gives the absorbance of a 1 mg/mL solution, $E^{0.1\%}$:

where $MW$ is in daltons. This is the form most lab protocols and spectrophotometer presets use, and it appears in the Abs 0.1% reduced and Abs 0.1% oxidized columns. The calculator computes the molecular weight from the same sequence, so both numbers describe the same construct. The values below come from running each sequence through the calculator with the oxidized assumption:

The lysozyme and BSA predictions sit close to the measured values most labs use, about 2.64 and 0.66. To convert a published $E^{1\%}$ into a molar coefficient, multiply by molecular weight and divide by 10: $\varepsilon = E^{1\%} \times MW / 10$.

Molecular weight here is the polypeptide alone. Glycans, lipids and other modifications add mass but no absorbance at 280 nm, so the mg/mL figure is protein mass, not the mass of the whole glycoprotein. For a profile that adds pI, amino acid composition and stability indices to the same coefficients, use Protein Parameters.

Antibody extinction coefficient

An IgG weighs about 150 kDa, and its exact tryptophan and tyrosine content depends on the variable regions, so each antibody has its own coefficient. Many spectrophotometers ship a generic IgG preset of $E^{1\%} = 13.7$, an $E^{0.1\%}$ of 1.37. For trastuzumab the sequence gives 1.484, so the generic preset would report a concentration 8% higher than the sequence-based value.

The calculator treats each FASTA record as a separate protein, so heavy and light chains entered as two records come back as two rows. To get values for the assembled antibody, join the chains into one record.

Two heavy plus two light chains give the whole IgG (215,380 $\text{M}^{-1}\,\text{cm}^{-1}$ for trastuzumab). One heavy plus one light chain gives the same $E^{0.1\%}$ and mg/mL but a µM value twice the IgG molarity. An intact IgG1 has 32 cysteines in 16 disulfides, so the oxidized coefficient applies.

How to determine an extinction coefficient

A sequence prediction is an average over many proteins. Pace and colleagues concluded that measuring is better than predicting when the number has to be right, and they found the Edelhoch method the best way to measure:

1. Prepare two samples from the same protein stock at the same dilution, one in your native buffer and one in 6 M guanidine hydrochloride.
2. Measure $A_{280}$ of both against matching blanks.
3. Calculate the denatured coefficient from the Trp, Tyr and Cys counts in the result table, using the Edelhoch values: $\varepsilon_{\text{denatured}} = 5690\,n_{Trp} + 1280\,n_{Tyr} + 120\,n_{cystine}$.
4. Correct it to the native protein: $\varepsilon_{\text{native}} = \varepsilon_{\text{denatured}} \times A_{\text{native}} / A_{\text{denatured}}$.

Unfolding works because it puts every chromophore in a solvent where its absorbance is known, removing the folded environment that makes prediction uncertain. Dry weight, nitrogen determination and amino acid analysis measure concentration absolutely, but they take more protein and more time.

When A280 misleads

A280 measures every molecule in the cuvette that absorbs at 280 nm, and the calculator assumes all of that absorbance comes from the sequence you pasted. The common ways the assumption fails:

Nucleic acid contamination deserves a number. RNA reads about 1 at 260 nm at 40 µg/mL, which puts its 280 nm absorbance near 12.5 per mg/mL. By that arithmetic, 10 µg/mL of RNA in a 1 mg/mL BSA sample adds about 0.12 to an expected 0.65, and the calculated concentration comes out roughly 19% high.

Measure within the linear range the instrument manufacturer specifies, and dilute a concentrated sample rather than trusting a reading above it.

Input

Several FASTA records give one result row each:

>Protein1
MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGE
>Protein2
GIVEQCCTSICSLYQLENYCN

CSV input needs a header row:

id,sequence
p1,MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGE
p2,GIVEQCCTSICSLYQLENYCN

Rejected characters stop the run with an error that names them, instead of being removed silently and shifting the counts.

Settings

Specified disulfide coefficient only takes effect when Cysteine assumption is Specified disulfide count; otherwise the calculator falls back to the oxidized coefficient.

Results

Each sequence returns one row:

Optional columns appear only when they have data:

Warnings appear above the table:

Reduced and oxidized values are assumptions about the sample, not structural predictions. The calculator does not infer which cysteines are bonded.

Frequently asked questions

How do I calculate protein concentration from A280?

Divide the absorbance by the molar extinction coefficient and the path length, then multiply by any dilution factor: $c = A_{280} / (\varepsilon \cdot l) \times d$. Multiply the molar result by molecular weight for mg/mL. Entering A280, path length and dilution here does both steps from the sequence.

What are the units of the extinction coefficient?

The molar extinction coefficient is in $\text{M}^{-1}\,\text{cm}^{-1}$, the same as $\text{L}\,\text{mol}^{-1}\,\text{cm}^{-1}$. The mass forms are $E^{0.1\%}$ in $(\text{mg/mL})^{-1}\,\text{cm}^{-1}$ and $E^{1\%}$ in $(\text{g/100 mL})^{-1}\,\text{cm}^{-1}$.

What is the extinction coefficient of BSA?

From the mature sequence, BSA has a molar coefficient of 42,925 $\text{M}^{-1}\,\text{cm}^{-1}$ with its 17 disulfides and 40,800 reduced. With a mass of 66,433 Da, that is an $E^{0.1\%}$ of 0.646, close to the measured value of about 0.66 that most labs use.

Should I use the reduced or oxidized extinction coefficient?

Match the state of the sample. Use reduced for cytoplasmic proteins and anything in a reducing agent, oxidized for secreted proteins and antibodies with native disulfides. For proteins with many tryptophans the two differ by about 1%; for cysteine-rich proteins with few aromatics the difference can exceed 5%.

Why does the calculator warn about proteins without tryptophan?

Tryptophan dominates absorbance at 280 nm, and its contribution is the most predictable. Without it the estimate depends on tyrosine and cystine, whose absorbance shifts more with their surroundings, so the predicted coefficient can be well off the measured one.

Can I calculate a DNA or RNA extinction coefficient?

Not with this calculator, which handles protein only. For oligonucleotides, the oligo analyzer reports the 260 nm extinction coefficient along with melting temperature and molecular weight.

References

• 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). doi:10.1002/pro.5560041120
• Gill SC, von Hippel PH. "Calculation of protein extinction coefficients from amino acid sequence data." Analytical Biochemistry 182, 319-326 (1989). doi:10.1016/0003-2697(89)90602-7
• Edelhoch H. "Spectroscopic determination of tryptophan and tyrosine in proteins." Biochemistry 6, 1948-1954 (1967).