Experimental Steps to Determine Protein Extinction Coefficient

The extinction coefficient of proteins is generally related to its absorbance at a specific wavelength (e.g., 280 nm). This is mainly due to some amino acid residues in proteins (such as tryptophan, tyrosine, and cysteine) having certain absorption characteristics at this wavelength.

 

Basic Steps for Determining the Protein Extinction Coefficient

1. Sample Submission Requirements

(1) Prepare a pure protein solution, ensuring it does not contain any other substances (such as salts, chelating agents, etc.) that may interfere with the measurement.

(2) Choose an appropriate buffer solution to maintain the stability of the protein.

 

2. Establish a Concentration Gradient

Prepare a series of protein solution samples with different concentrations. Ensure that the concentrations of these samples are in the same range so that their absorbance values ​​are within the linear range of the spectrophotometer.

 

3. Absorbance Measurement

(1) Measure the absorbance of each sample at a specific wavelength (e.g., 280 nm) using a photometer or spectrophotometer.

(2) Measure the absorbance of a blank sample (containing only the buffer solution, no protein) for later correction.

 

4. Data Analysis

(1) Use the measured absorbance and known protein concentration to establish a relationship curve between absorbance and concentration.

(2) This relationship should be linear, and the slope is the extinction coefficient.

 

5. Calculate the Extinction Coefficient

(1) According to Beer's law: A=ε×c×l

(2) Here, A is the absorbance, c is the concentration of the protein, and l is the optical path length (usually 1 cm). From the slope of the above relationship curve, you can directly obtain the extinction coefficient ε.

 

This method provides an intuitive, experimental way to measure the extinction coefficient of proteins. It is particularly important for proteins that do not have a known extinction coefficient or whose amino acid sequence is known but want to verify whether the extinction coefficient changes under experimental conditions.