Detection of Proteins Using SDS-PAGE

SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) is a powerful technique for protein separation. It is widely used in biological research to analyze the composition of protein mixtures, determine the relative molecular mass of proteins, and isolate specific proteins for further study.

 

SDS-PAGE utilizes the combined actions of SDS and polyacrylamide gel. SDS is an anionic detergent that disrupts the secondary and tertiary structures of proteins, denaturing them and imparting a uniform negative charge. This ensures that the migration rate of proteins in an electric field is primarily determined by their size. Polyacrylamide gel acts as a molecular sieve. The concentration of polyacrylamide gel determines its resolving power. Typical concentrations range from 5% to 20%. Lower concentrations are suitable for separating large proteins, while higher concentrations are ideal for small proteins. By adjusting the gel concentration, researchers can optimize the separation of specific proteins.

 

Electrophoresis Process

The electrophoresis process includes four steps: sample preparation, gel preparation, electrophoresis running, and staining detection.

 

1. Sample Preparation

Protein samples are mixed with SDS and a reducing agent (such as β-mercaptoethanol or DTT) and heated to achieve thorough denaturation.

 

2. Gel Preparation

The appropriate concentration of resolving gel and stacking gel is selected based on the target protein size. The resolving gel is used for actual protein separation, while the stacking gel is used to concentrate the samples.

 

3. Electrophoresis Running

Samples are loaded into gel wells and migrate from the cathode to the anode under the influence of an electric field. Smaller proteins migrate faster, while larger proteins migrate more slowly.

 

4. Staining Detection

After the run, the gel is stained (e.g., with Coomassie Brilliant Blue or silver stain) to visualize protein bands. Proteins of different sizes form bands at different positions, facilitating analysis.

 

Data Analysis

By comparing the results with known molecular weight standards, researchers can estimate the relative molecular mass of proteins in the samples. Additionally, the intensity of the bands can indicate the relative abundance of proteins.

 

Application Fields

SDS-PAGE is widely used in protein purification, proteomics research, and protein-protein interaction studies. In medical research, it is used to diagnose certain diseases (such as muscular dystrophy) by identifying characteristic protein deficiencies or abnormal expressions.