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. How do I do that?
The extinction coefficient of a protein can be determined 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 from the number of A280 absorbing residues (Trp, Tyr, Cystine – disulfide bonds).
However, the actual molar extinction coefficient depends on the buffer and 3-dimensional structure of the protein. For accurate protein concentration measurements by A280nm measurement I 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.
Suggested three-step procedure
If you don’t want to experiment with this yourself, Alphalyse also offers a great service to help you determine the ext. coefficient of your protein. The analysis is a three-step procedure:
- First we measure the protein concentration by quantitative amino acid analysis (AAA) in triplicate
- Then we determine the UV absorbance at 280 nm at different dilutions of the protein
- Finally, we calculate the absorptivity constant and molar extinction coefficient from the slope of the A280 curve.
I prefer to combine this analysis with an accurate protein quantification using triplicate amino acid analysis. This involves hydrolyzation of the sample in HCl in triplicate followed by amino acid analysis using a Biochrom +30 instrument. The analysis very accurately determines the protein amount. The molar concentration can then be determined using the amino acid composition of the protein.
Based on the results from the AAA analysis, we prepare a dilution series covering the highest possible concentration and a 50-fold dilution – all measured at 280 nm. From these data, we derive a linear curve (see example at the top of this page), which we use in determination of the extinction coefficient.
The analysis is typically within 10% accuracy. We 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 we make the extinction coefficient determination in the buffer system you are going to use in your lab. The sample should be as concentrated as possible. This ensures that we get an extinction coefficient that covers as large a concentration range as possible.
For accurate amino acid analysis, you should avoid material in the buffer that can polymerize during the acidic hydrolysis, taking place at 110°C (e.g. sugars).
UV measurement at 280 nm and triplicate amino acid analysis
Alphalyse combines UV measurement at 280 nm with triplicate amino acid analysis. The combination results in a very accurate concentration determination. Find more information about Molar Extinction Coefficient determination on the Alphalyse website.