Workflow of SDS-PAGE Based Protein Separation

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) is a widely utilized technique for separating proteins based on their electrophoretic mobility through a polyacrylamide gel matrix. The following outlines the detailed workflow of SDS-PAGE protein separation.

 

Sample Preparation

Initially, proteins in the sample must be treated to ensure effective separation. The steps are as follows:

 

1. Protein Extraction

Extract proteins from cells or tissues, and remove cell debris and other impurities by centrifugation or other separation methods.

 

2. Protein Denaturation

Add SDS (sodium dodecyl sulfate) and reducing agents (such as β-mercaptoethanol), and heat the mixture to denature the proteins. SDS binds to proteins, imparting a negative charge and disrupting their native structure.

 

3. Sample Buffer

Mix the denatured proteins with sample buffer, which typically contains glycerol (to increase sample density) and bromophenol blue (for visualization during electrophoresis).

 

Gel Preparation

The polyacrylamide gel used in SDS-PAGE consists of two parts: the resolving gel and the stacking gel.

 

1. Resolving Gel

This gel, with a higher concentration of acrylamide, separates proteins of different sizes. The concentration of the resolving gel (usually between 7.5% and 20%) is adjusted based on the size of the target proteins. Smaller proteins separate more effectively in higher concentration gels.

 

2. Stacking Gel

This gel, with a lower concentration of acrylamide (usually 4%), concentrates the proteins into narrow bands, improving the separation efficiency.

 

Sample Loading

Load the prepared protein samples into the wells of the gel. Use a micropipette to carefully inject the sample into each well to prevent diffusion or leakage.

 

Electrophoresis

Conduct the electrophoresis process in an electrophoresis apparatus:

 

1. Voltage Setting

Apply an initial low voltage (e.g., 60-80 volts) to focus the proteins in the stacking gel. Then increase the voltage (e.g., 120-200 volts) to separate the proteins in the resolving gel.

 

2. Electrophoresis Buffer

Add electrophoresis buffer, usually Tris-Glycine buffer, to the electrophoresis tank to ensure even electric field distribution.

 

Protein Staining

After electrophoresis, stain the proteins for visualization. Common staining methods include:

 

1. Coomassie Brilliant Blue Staining

Immerse the gel in Coomassie Brilliant Blue staining solution, and the protein bands will appear blue.

 

2. Silver Staining

Silver staining has higher sensitivity and is suitable for detecting low-concentration proteins.

 

Result Analysis

Image the stained gel using an optical scanner or camera. By comparing the migration distance of protein bands with known molecular weight standards, determine the molecular weight of the proteins in the sample.