Principle of SDS-PAGE Based Protein Separation

Proteins are essential biological macromolecules in living organisms, and studying their structure and function is crucial for understanding biological phenomena. In biological research, protein separation and identification are fundamental steps, and SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) is a widely used technique for efficient protein separation. This article will detail the principles of protein separation by SDS-PAGE.

 

Basic Principles of SDS-PAGE

SDS-PAGE is an electrophoresis-based method for separating proteins, relying on the combined use of SDS and polyacrylamide gel. The basic principles include the following aspects:

 

1. Role of SDS

SDS is an anionic detergent that binds to and denatures protein molecules. Specifically, SDS molecules interact with the hydrophobic regions of proteins, causing them to unfold into linear chains. Additionally, each SDS molecule provides a negative charge, ensuring that proteins migrate in an electric field with similar charge densities. This process ensures that protein separation depends solely on molecular weight and is not influenced by the proteins' native shape or charge.

 

2. Role of Polyacrylamide Gel

Polyacrylamide gel is formed by the polymerization of acrylamide and the cross-linker bisacrylamide, creating a three-dimensional network structure. The pore size of the gel can be adjusted by varying the ratios of acrylamide and the cross-linker, making it suitable for separating proteins of different molecular weights. Smaller protein molecules can pass through the gel pores more quickly, while larger proteins pass through more slowly. This allows proteins to be separated based on their molecular weight under the influence of an electric field.

 

3. Electrophoresis Process

During SDS-PAGE, samples are first mixed with SDS and boiled to ensure complete denaturation and binding of proteins with SDS. The treated samples are then loaded into wells of a polyacrylamide gel. Under the influence of an electric field, the negatively charged protein molecules migrate from the cathode to the anode. Due to the sieving effect of the polyacrylamide gel, proteins are separated according to their molecular weight.

 

Factors Affecting SDS-PAGE Separation

1. Gel Concentration

The concentration of acrylamide and the cross-linker in the gel determines the pore size, which is crucial for optimal separation of proteins of different molecular weights. Generally, high-concentration gels are used for low-molecular-weight proteins, while low-concentration gels are used for high-molecular-weight proteins.

 

2. Voltage and Current

The voltage and current applied during electrophoresis directly affect the migration speed and separation efficiency of proteins. Typically, lower voltage and current yield better separation results but require longer electrophoresis time.

 

3. Buffer Systems

The choice of electrophoresis buffer also affects the outcome of SDS-PAGE. Common buffer systems include Tris-Glycine and Tris-Tricine, each suitable for separating proteins within different molecular weight ranges.

 

In summary, SDS-PAGE is an efficient and widely used protein separation technique based on SDS denaturation and polyacrylamide gel sieving effects. By optimizing experimental conditions, high-resolution protein separation can be achieved, providing critical technical support for biological research.