Steps for Protein Determination Using Circular Dichroism

Circular Dichroism (CD) is a spectroscopic technique based on electromagnetic waves that can analyze the structural features of proteins by measuring the difference in absorption of left and right circularly polarized light. It is currently widely used in determining the secondary structure of proteins, and is a fast, simple, and relatively accurate method for studying the conformation of proteins in dilute solutions.

 

Analysis Workflow

1. Sample Preparation

High concentration protein solution is obtained from the sample by purification techniques such as chromatography or electrophoresis. In this process, the sample should be placed in an appropriate buffer solution and the concentration adjusted to 0.1~1.0 mg/mL.

 

2. Instrument Calibration

Before using the instrument, calibration should be done, including checking the stability of the light source, the accuracy of the wavelength and the sensitivity of the detector. At the same time, the cleanliness of the optical system should be maintained to ensure the reliability of the measurement results.

 

3. Background Measurement

To eliminate the impact of substances absorbed in the buffer solution on the experimental results, the CD signal of the buffer solution should be measured before adding the sample to obtain a baseline, which is then subtracted from the protein signal in the subsequent data processing.

 

4. Sample Measurement

After loading the protein sample into a quartz cuvette, it is placed in the instrument, and the appropriate wavelength range (190~250 nm) and temperature (20℃) are set before starting the measurement. During the measurement process, the temperature should be kept constant to avoid the impact of temperature fluctuations on the results.

 

5. Data Processing

The background signal is subtracted from the CD signal of the sample to get the net CD spectrum. By calculating and comparing the values of absorption differences, the circular dichroism spectrum of the protein is obtained.

 

6. Data Analysis

(1) Qualitative Analysis: By comparing the CD spectrum of the sample with known spectral features, the secondary structure content of the sample can be inferred, such as the amount and position of α-helices and β-folds, and local conformational changes in proteins.

(2) Quantitative Analysis: Special software and algorithms such as SELCON3, CONTIN or CDPro are used to calculate the proportions of various secondary structures based on CD spectrum data.

 

In addition, by measuring the CD spectrum of the sample under different conditions, such as different pH, temperature or ion strength, the conformational stability and conformational changes of the sample can be studied.