Workflow of 2D Gel Electrophoresis Image Analysis

Two-dimensional gel electrophoresis (2-DE) is a high-resolution protein separation technique widely used in proteomics research. It combines isoelectric focusing (IEF) and SDS-PAGE electrophoresis, allowing the separation of complex protein mixtures in two-dimensional space. The analysis of 2-DE images is a crucial part of this technique, enabling quantitative and qualitative understanding of protein expression in samples. The following is the workflow of 2-DE image analysis.

 

1. Sample Preparation

Sample preparation is the first step in 2-DE and usually involves processes of protein extraction, purification, and concentration. High-quality samples are key to successful electrophoresis. Common protein extraction methods include mechanical disruption, sonication, and chemical lysis.

 

2. First-Dimensional Electrophoresis: Isoelectric Focusing

Isoelectric focusing is the first-dimensional separation process of 2-DE. Proteins move in a pH gradient until they reach their isoelectric point (pI), where the net charge of the protein is zero, and they stop moving. IEF is usually performed on polyacrylamide gel strips (IPG strips).

 

3. Second-Dimensional Electrophoresis: SDS-PAGE

The second-dimensional separation utilizes SDS-polyacrylamide gel electrophoresis (SDS-PAGE). After IEF separation, the IPG strips are equilibrated in an SDS solution and placed on SDS-PAGE gels for further separation by molecular weight.

 

4. Image Acquisition

After 2-DE separation, the gels need to be stained to visualize the protein spots. Common staining methods include Coomassie Brilliant Blue, silver staining, and fluorescent staining. The stained gels are then scanned or imaged using an imaging system.

 

5. Image Preprocessing

The acquired 2-DE images require preprocessing to enhance image quality and prepare for subsequent analysis. Preprocessing steps include image correction, background subtraction, and noise removal.

 

6. Spot Detection

Spot detection is a key step in image analysis. Specialized software is used to identify and detect protein spots in the 2-DE images. Commonly used software includes ImageMaster, PDQuest, and Progenesis.

 

7. Spot Matching

Due to variations between samples, spot matching is needed to align spots in different images that correspond to the same protein. This allows for comparative analysis across samples.

 

8. Quantitative Analysis

Based on spot matching, quantitative analysis is performed. This usually involves comparing the intensity or area of spots to assess changes in the relative abundance of proteins.

 

9. Data Analysis and Interpretation

The final step is data analysis and interpretation. Through statistical analysis of quantitative results, proteins with significant expression differences under different conditions are identified. Further identification and functional annotation of proteins are performed using mass spectrometry and database searches.