Principle of 2D Gel Electrophoresis

Two-Dimensional Gel Electrophoresis (2-DE) is a high-resolution protein separation technique widely used in proteomics research. This method combines isoelectric focusing (IEF) and SDS-polyacrylamide gel electrophoresis (SDS-PAGE), enabling the efficient separation of complex protein mixtures based on their isoelectric points (pI) and molecular weights (MW).

 

Isoelectric Focusing (IEF)

Isoelectric focusing is the first step in 2-DE. IEF utilizes the property of proteins where they become electrically neutral at their isoelectric point, which is specific to each protein at a particular pH. During IEF, protein samples are loaded into a polyacrylamide gel containing a pH gradient. When an electric field is applied, proteins migrate within the gel until they reach their isoelectric point. At this pH, the proteins have no net charge and thus stop moving. This method allows for the high-resolution separation of proteins based on their isoelectric points, distinguishing proteins with very small pI differences.

 

SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

Following IEF, the second step is SDS-PAGE. This process involves the use of sodium dodecyl sulfate (SDS) to bind proteins, conferring a uniform negative charge, and separates them based on molecular weight. The IEF gel strip is placed on top of the SDS-PAGE gel, and proteins migrate downwards under the influence of an electric field. Since SDS binding eliminates the original structural and charge differences between proteins, SDS-PAGE primarily separates proteins according to their molecular weights. Smaller proteins migrate faster, while larger proteins migrate slower.

 

Result Analysis

Upon completion of 2-DE, the distribution of proteins on the gel can be visualized using staining methods such as silver staining or Coomassie Brilliant Blue staining. Each protein's position on the two-dimensional gel is determined by its isoelectric point and molecular weight, resulting in a detailed map of the protein mixture. Techniques like mass spectrometry can further identify these proteins and elucidate their functions.

 

Two-dimensional gel electrophoresis has broad applications in proteomics, molecular biology, and medicine. It can reveal changes in protein expression under various physiological and pathological conditions, offering critical insights for disease diagnosis and treatment.