What is the Difference Between Stacking Gel and Separating Gel?

Stacking gel and separating gel are two types of polyacrylamide gels used in SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) to achieve better separation of protein molecules in a given sample. The main differences between these two gels are:

• Purpose: The purpose of the stacking gel is to line up all the protein samples loaded on the gel so that they can enter the resolving gel at the same time. The resolving gel, also known as the separating gel, is designed to separate the proteins based on their molecular weight.
• pH: The stacking gel has a lower pH (6.8) than the resolving gel (8.8).
• Polyacrylamide Content: The polyacrylamide content in the stacking gel (usually around 4%) is lower than that in the resolving gel (around 10-15%).
• Pore Size: The stacking gel has larger pore sizes, while the resolving gel has smaller pore sizes.
• Resolution: The stacking gel provides better resolution, while the resolving gel offers poorer resolution.

In summary, stacking gel and separating gel are two essential components of SDS-PAGE, with the stacking gel serving to align protein samples and the separating gel working to resolve them based on their molecular weight. These differences in pH, polyacrylamide content, and pore size contribute to the unique functions of each gel type.

Comparative Table: Stacking Gel vs Separating Gel

The main difference between stacking gel and separating gel lies in their pH, polyacrylamide content, pore size, and purpose. Here is a comparison table highlighting the differences:

Stacking gel is a low concentrated polyacrylamide gel placed on top of a more concentrated separating gel in SDS-PAGE technique. The purpose of the stacking gel is to concentrate all the different sized proteins into a compact area, ensuring that most of the proteins enter the denser resolving gel simultaneously before they begin to separate. This results in clearer and better-separated bands for visualization and analysis.

On the other hand, the separating gel, also known as the running gel or resolving gel, is a highly concentrated polyacrylamide gel placed at the bottom of the container used for gel electrophoresis. Its purpose is to separate the proteins based on their molecular weight, with higher molecular weight proteins moving more slowly through the porous gel.