Difference gel electrophoresis

Difference Gel Electrophoresis (DIGE) is a powerful analytical technique used in proteomics to compare protein abundance between different biological samples. This method allows for the simultaneous separation and quantification of proteins from multiple samples on the same polyacrylamide gel, thereby reducing gel-to-gel variability and increasing the accuracy of protein expression analysis.

Principle

DIGE is based on the principle of two-dimensional gel electrophoresis (2D-GE), which separates proteins according to their isoelectric point (pI) and molecular weight. In DIGE, proteins from different samples are labeled with distinct fluorescent dyes before being mixed and run on the same gel. This allows for direct comparison of protein expression levels across samples.

Fluorescent Labeling

The key innovation in DIGE is the use of fluorescent dyes to label proteins. Typically, CyDye DIGE Fluor minimal dyes such as Cy2, Cy3, and Cy5 are used. These dyes are charge-matched and mass-matched, ensuring that they do not alter the migration of proteins during electrophoresis. Each sample is labeled with a different dye, and a pooled internal standard is often labeled with a third dye to facilitate accurate quantification.

Sample Preparation

Sample preparation for DIGE involves several steps:

• Protein Extraction: Proteins are extracted from biological samples using appropriate lysis buffers that maintain protein solubility and integrity.
• Protein Quantification: Accurate quantification of protein concentration is essential to ensure equal loading of samples.
• Labeling: Proteins are labeled with CyDyes according to the manufacturer's protocol, typically at a ratio of 400 pmol dye per 50 µg protein.

Two-Dimensional Gel Electrophoresis

In the first dimension, proteins are separated by isoelectric focusing (IEF), which resolves proteins based on their pI. In the second dimension, proteins are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which resolves proteins based on their molecular weight.

Image Acquisition and Analysis

After electrophoresis, the gel is scanned using a fluorescence scanner to detect the labeled proteins. Each dye is excited at its specific wavelength, and the emitted fluorescence is captured to generate images for each sample. These images are then analyzed using specialized software to quantify differences in protein expression.

Applications

DIGE is widely used in biomedical research for:

• Biomarker Discovery: Identifying proteins that are differentially expressed in disease states.
• Comparative Proteomics: Comparing protein expression profiles between different conditions or treatments.
• Functional Proteomics: Studying changes in protein expression in response to cell signaling or environmental stimuli.

Advantages

DIGE offers several advantages over traditional 2D-GE:

• Reduced Variability: By running samples on the same gel, DIGE minimizes gel-to-gel variability.
• Increased Sensitivity: The use of fluorescent dyes allows for the detection of low-abundance proteins.
• Quantitative Analysis: The inclusion of an internal standard enables accurate quantification of protein expression changes.

Limitations

Despite its advantages, DIGE has some limitations:

• Complexity: The technique requires careful optimization of labeling and electrophoresis conditions.
• Cost: The fluorescent dyes and specialized equipment can be expensive.
• Protein Coverage: Like all gel-based methods, DIGE may not resolve very high or very low molecular weight proteins effectively.