Workflow of 2D Gel Electrophoresis

Two-Dimensional Gel Electrophoresis (2-DE) is a robust technique widely employed in proteomics to separate proteins based on their isoelectric point (pI) and molecular weight. This article provides a detailed workflow of the 2-DE process.

 

Sample Preparation

Effective sample preparation is critical for 2-DE, as it impacts the quality of protein separation and the reliability of the results. Proteins are extracted and purified from samples while minimizing degradation. Typically, cell lysates or tissue homogenates are used, followed by centrifugation to remove debris and insoluble materials. Protease inhibitors are included to preserve protein integrity.

 

Isoelectric Focusing (IEF)

IEF is the first dimension in 2-DE, separating proteins based on their pI in a pH gradient under an electric field. This step is performed on Immobilized pH Gradient (IPG) strips that are pre-soaked in a specific pH gradient buffer.

 

1. IPG Strip Rehydration

IPG strips are rehydrated with a buffer containing the sample for 12-16 hours, ensuring thorough absorption of the sample.

 

2. IEF Execution

Post-rehydration, the IPG strips are placed in an IEF apparatus, where voltage is gradually increased over 10-20 hours, depending on the sample and equipment specifics.

 

SDS-PAGE

Following IEF, proteins are further separated by molecular weight in the second dimension using SDS-PAGE. SDS, an anionic detergent, denatures proteins and imparts a uniform negative charge, allowing separation by size.

 

1. IPG Strip Equilibration

Equilibrate IPG strips in a buffer containing urea, DTT, and SDS for 15-30 minutes to ensure complete protein denaturation.

 

2. SDS-PAGE Execution

Place the equilibrated IPG strips on top of the SDS-PAGE gel, and apply an electric field to facilitate protein separation. Conditions are adjusted based on gel concentration and protein size, typically running for 2-4 hours.

 

Protein Detection

Post-2-DE, protein detection is necessary. Methods like Coomassie Brilliant Blue staining, silver staining, and fluorescence staining are employed based on protein abundance and study requirements.

 

1. Staining

The gel is immersed in a staining solution for 1-2 hours, depending on the chosen method.

 

2. Destaining

Excess dye is removed using a destaining solution for 1-2 hours until the background is clear.

 

Data Analysis

Finally, the separated protein spots are analyzed. Gel images are captured using a gel imaging system, and specialized software identifies and quantifies the protein spots. Comparative analysis of protein expression profiles between different samples enables the identification of differentially expressed proteins, aiding further functional studies.