Mechanism of 2D-Electrophoresis

Two-Dimensional Electrophoresis (2-DE) is a widely utilized separation technique in proteomics research. By leveraging the differences in proteins' isoelectric points (pI) and molecular weights, 2-DE achieves high-resolution separation of complex protein mixtures through two sequential, orthogonal electrophoresis processes.

 

The fundamental principle of 2-DE is the sequential separation of protein samples using two distinct electrophoresis processes: Isoelectric Focusing (IEF) and SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis). These processes separate proteins based on their isoelectric points and molecular weights, respectively.

 

1. Isoelectric Focusing (IEF)

In the first step, IEF, the protein sample is separated in a polyacrylamide gel containing a pH gradient. Under the influence of an electric field, protein molecules migrate to their isoelectric points, where the pH equals their pI and they carry no net charge, thereby halting their migration. This separation is based on the proteins' isoelectric points.

 

2. SDS-PAGE

In the second step, SDS-PAGE, the proteins separated by IEF are further resolved in a polyacrylamide gel containing SDS (Sodium Dodecyl Sulfate). SDS uniformly imparts a negative charge to the proteins, allowing their separation solely based on molecular weight during electrophoresis. Smaller proteins migrate faster, while larger proteins migrate slower.

 

Steps

The typical workflow of 2-DE includes the following critical steps:

 

1. Sample Preparation

Sample preparation is pivotal for the success of 2-DE. It generally encompasses cell or tissue lysis, protein extraction and purification, and preparation of the sample solution.

 

2. Isoelectric Focusing

The sample solution is loaded into an IEF gel with a pH gradient, and isoelectric focusing is performed under an electric field. This process usually spans several hours until the proteins are completely separated according to their isoelectric points.

 

3. SDS-PAGE

After IEF, the gel strip is transferred to an SDS-PAGE gel, where the second electrophoresis step is performed under an electric field, further separating proteins based on their molecular weights.

 

4. Protein Staining and Detection

Following electrophoresis, the proteins in the gel need to be visualized using staining methods such as Coomassie Brilliant Blue or silver staining. Subsequent identification and analysis of the proteins can be conducted using techniques like mass spectrometry or western blotting.

 

Applications

2-DE has significant applications in biological research, particularly in proteomics. It can be used for:

 

1. Protein Expression Profiling

Comparing protein expression differences under various conditions in cells or tissues.

 

2. Protein Modification Studies

Detecting post-translational modifications such as phosphorylation and glycosylation.

 

3. Disease Biomarker Discovery

Identifying potential disease biomarkers by comparing protein expression profiles between healthy and diseased tissues.