Workflow of 2D-DIGE-Based Protein Quantification

Two-Dimensional Difference Gel Electrophoresis (2D-DIGE) is a powerful proteomics technique for the simultaneous separation and quantification of proteins from different biological samples. This technique significantly improves the accuracy and sensitivity of protein quantification by labeling and separating multiple samples on the same gel.

 

Workflow

1. Sample Preparation

The success of 2D-DIGE hinges on meticulous sample preparation. First, suitable biological samples, such as tissues or cells, are selected, lysed, and the total protein extracted. The protein samples are quantified using methods like BCA or Bradford to ensure consistent protein concentration across samples. They are then reduced and alkylated to disrupt disulfide bonds and prevent nonspecific protein interactions.

 

2. Dye Labeling

Following preparation, samples are labeled with CyDye dyes—typically Cy3, Cy5, and Cy2 for different samples and internal standards. Each sample is combined with the appropriate dye and incubated on ice in the dark. After incubation, a stop buffer is added to halt the labeling reaction. This step ensures that fluorescence intensity remains comparable across wavelengths.

 

3. Isoelectric Focusing (IEF)

Labeled protein mixtures are loaded onto isoelectric focusing strips. During IEF, proteins are separated based on their isoelectric points (pI). As electrophoresis progresses, proteins migrate along the pH gradient until they reach the position where their net charge is zero, forming distinct bands. This process usually takes several hours and requires stringent control of conditions.

 

4. SDS-PAGE

Post-IEF, the strips undergo equilibration to impart a uniform negative charge to the proteins, and are then placed on an SDS-PAGE gel. This step further separates the proteins by molecular weight. SDS-PAGE requires precise control to ensure the resolution and reproducibility of protein bands.

 

5. Gel Imaging and Data Analysis

Upon completion of electrophoresis, images of the fluorescently labeled proteins are captured using a specialized imaging system. Software is then employed to detect, match, and quantify protein spots. The relative abundance of each protein is calculated by comparing the fluorescence intensity of corresponding spots across different samples. Data analysis involves background subtraction, spot matching, and statistical analysis to identify proteins with significant expression differences.

 

6. Protein Identification

Finally, protein spots showing significant expression changes are selected for mass spectrometry analysis. Mass spectrometry provides precise molecular weight and sequence data, facilitating further functional studies.

 

2D-DIGE-based protein quantification remains a critical tool in proteomics research due to its high resolution and accuracy. Its standardized workflow offers a reliable foundation for protein separation, quantification, and identification.