Determine the Molar Extinction Coefficient of protein in 3 small steps

Extinction coefficient protein concentration
Nov 15. 2016


I have a purified protein that I would like to quantify accurately in my lab using 280 nm UV measurement (A280). To do so, I need to determine the molar extinction coefficient of the protein in my buffer system. What is the best way to do that?


It is possible to determine the molar extinction coefficient (also known as the molar attenuation coefficient) of a protein experimentally. You do this by A280 measurements of a dilution series of the protein in known concentrations. An extinction coefficient can also be predicted by a theoretical calculation. This is based on the number of A280 absorbing residues (Trp, Tyr, Cystine – disulfide bonds) [1].

However, the actual molar extinction coefficient depends on the buffer and 3-dimensional structure of the protein. For accurate protein concentration determination by A280nm measurement we therefore recommend that you determine the extinction coefficient experimentally in the buffer you are going to use in your lab, for example in PBS buffer [2].

Suggested three-step procedure to determine the extinction coefficient

To determine the extinction/attenuation coefficient, you will need access to an amino acid analyzer and also a spectrophotometer. The analysis is then a three-step procedure:

  1. Measure the exact protein concentration
    First hydrolyze the sample in HCl in triplicate and measure the protein concentration by quantitative amino acid analysis (AAA), e.g. using a Biochrom 30+ instrument, which performs ion exchange chromatography and post column derivatization with Ninhydrin.
    Avoid material in the buffer that can polymerize during the acidic hydrolysis, taking place at 110°C (e.g. sugars)

    Next, determine the molar concentration using the amino acid composition of the protein [1,2].

  2. Determine the UV absorbance
    Based on the results from the AAA analysis, prepare a dilution series covering the highest possible concentration and a 50-fold dilution – and measure the UV absorbance at 280 nm.

    Now, derive a linear curve from the absorbance as a function of the concentration (see example at the top of this page).

  3. Calculate the molar extinction coefficient
    Finally, using the Beer-Lambert law, calculate the absorptivity constant and molar extinction coefficient from the slope of the A280 curve and the molecular weight of the protein.

The analysis is typically within 10% accuracy. You can ensure this quality by running a BSA sample (NIST standard) along with your samples.

Things to consider when determining the extinction coefficient of proteins

It is very important that you make the extinction coefficient determination in the buffer system you are going to use in your lab. Also, the sample should be as concentrated as possible. This ensures that you get an extinction coefficient that covers as large a concentration range as possible [1, 2].

UV measurement at 280 nm and triplicate amino acid analysis

If you do not want to experiment with this yourself, Alphalyse also offers a great service to help you determine the ext. coefficient of your protein.

Alphalyse combines UV measurement at 280 nm with accurate amino acid analysis. Find more information about Molar Extinction Coefficient determination on the Alphalyse website.


[1]          Pace et al: How to measure and predict the molar absorption coefficient of a protein“, Protein Science1995

[2]          Gill et al: Calculation of protein extinction coefficients from amino acid sequence data“, Analytical Biochemistry1989

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