What is the Difference Between Stacking Gel and Separating Gel

Gel electrophoresis is a fundamental technique in molecular biology, enabling the separation and analysis of biomolecules. Stacking gel and separating gel are two critical components of this process. While both are made from polyacrylamide and used as a buffer system for electrophoresis, they serve distinct functions in the precise orchestration of molecule separation.

What is the difference between stacking gel and separating gel? Stacking gel helps concentrate and focus the sample into a tight band before it enters the separating gel, whereas separating gel separates molecules based on size during electrophoresis.

Key Areas Covered

1. What is a Stacking Gel 
      – Definition, Features, Applications
2. What is a Separating Gel 
      – Definition, Features, Applications 
3. Similarities Between Stacking Gel and Separating Gel
      – Outline of Common Features
4. Difference Between Stacking Gel and Separating Gel
      – Comparison of Key Differences
5. FAQ: Stacking Gel and Separating Gel
      – Frequently Asked Questions

Key Terms

Stacking Gel, Separating Gel

Difference Between Stacking Gel and Separating Gel - Comparison Summary

What is a Stacking Gel

Stacking gel is a crucial component in gel electrophoresis, a widely used technique for separating and analyzing biomolecules such as DNA, RNA, and proteins. The primary purpose of stacking gel is to focus and concentrate the sample before it enters the resolving gel, allowing for better resolution and more accurate analysis.

Typically composed of a lower concentration of acrylamide compared to the resolving gel, stacking gel creates a porous matrix through polymerization. This matrix acts as a molecular sieve, slowing down the movement of biomolecules and preventing them from diffusing too quickly. By temporarily trapping molecules, the stacking gel enables the formation of a sharp, compact sample band that migrates collectively into the resolving gel.

The composition of the stacking gel is carefully optimized to strike a balance between sieving properties and allowing efficient entry of the sample. Buffers and additives are incorporated to maintain a stable pH environment and provide suitable ionic conditions for electrophoresis. Tris-acetate and Tris-borate buffers are common choices, depending on the specific requirements of the experiment.

During electrophoresis, an electric current is applied across the gel, causing the charged biomolecules to migrate through the matrix. The stacking gel ensures that the sample components are concentrated into a narrow, well-defined band, which subsequently enters the resolving gel for separation based on size or charge. This process enhances the resolution of individual bands, allowing for accurate size estimation and analysis.

The choice of stacking gel concentration and thickness depends on the size range of the molecules. Higher-concentration stacking gels are suitable for smaller molecules, while lower concentrations accommodate larger ones. Additionally, varying the thickness of the stacking gel influences the degree of sample concentration and the sharpness of bands.

What is a Separating Gel

Separating gel is an essential component in the process of gel electrophoresis. Gel electrophoresis relies on the principle of migration through a gel matrix under the influence of an electric field. The gel typically used for separation in this technique is made of a polymer, commonly agarose or polyacrylamide. Separating gels in electrophoresis is distinct from stacking gels.

Polyacrylamide gels are preferable for high-resolution separation, particularly for proteins, due to their fine mesh-like structure. Agarose gels, on the other hand, are more suitable for separating larger fragments like DNA. The choice of gel type depends on the specific requirements of the experiment.

Differentiate Stacking Gel vs Separating Gel

The process of preparing the separating gel involves carefully measuring and mixing the gel components, which may include monomers, a cross-linking agent, a catalyst, and a buffer solution. Once the gel mixture is prepared, it is poured into a casting tray and allowed to polymerize. This results in the formation of a solid gel matrix with pores of varying sizes, creating a sieving effect during electrophoresis.

During the electrophoretic run, the sample is loaded into wells at one end of the gel, and an electric current is applied. The molecules in the sample move through the gel matrix at different rates based on their size and charge. Smaller molecules migrate more quickly and travel farther through the gel, while larger molecules lag behind.

 Similarities Between Stacking Gel and Separating Gel

  1. Both gels are made from polyacrylamide.
  2. They both use a buffer system to create an electric field for the movement of charged molecules through the gel.

Difference Between Stacking Gel and Separating Gel


  • A stacking gel is a gel with a lower concentration of polyacrylamide that concentrates and stacks the sample into a narrow band before it enters the separating gel in gel electrophoresis. On the other hand, a separating gel is a gel with a higher concentration of polyacrylamide that separates molecules based on size and charge in gel electrophoresis.


  • Stacking gel has a lower percentage of acrylamide, while separating gel has a higher percentage of acrylamide, resulting in a finer matrix for better resolution of separated proteins.


  • Stacking gel focuses on concentrating and stacking proteins into a tight band, preparing them for entry into the separating gel, while separating gel facilitates the separation of proteins based on their size as they migrate through the gel.

Pore Size

  • In stacking gel, larger pore size enables rapid movement of proteins, helping them concentrate at the interface with the separating gel. However, in separating gel, smaller pore size restricts the movement of proteins, promoting separation based on molecular weight.


In gel electrophoresis, the stacking gel and separating gel serve distinct purposes. The stacking gel, with larger pores and less acrylamide, concentrates molecules into a tight band, readying them for separation in the separating gel. Meanwhile, the separating gel, with a finer matrix, separates molecules based on size, revealing the sample’s composition. This interplay showcases the precise methodology of gel electrophoresis.

FAQ: Stacking Gel and Separating Gel

1. What is the purpose of a stacking gel?

The purpose of stacking the gel is to line up all the protein samples loaded on the gel. This makes it easier for them to enter the resolving gel at the same time.

2. What gel is used for DNA separation?

Agarose gel electrophoresis is a widely used technique due to its ability to separate DNA fragments based on size. This method is essential in various applications, including molecular biology research, genetic diagnostics, and DNA fingerprinting.

3. Why pH 6.8 is used in stacking gel?

pH 6.8 is used in the stacking gel of electrophoresis because it helps to ensure that the proteins remain negatively charged, allowing them to move through the gel matrix efficiently without being overly influenced by their charge.

4. Why is there a pH difference between stacking and separating gel?

The stacking gel, which has high porosity, is buffered with Tris-Cl buffer at pH 6.8. This pH level helps concentrate the sample into a narrow band before it enters the separating gel. Conversely, the separating gel, with a high percentage of acrylamide, is cast in Tris-Cl buffer at pH 8.8. This higher pH environment is optimal for separating molecules based on size and charge.


1. “Polyacrylamide Gel Electrophoresis – An Overview.” Science Direct.
2. “SDS-PAGE.” Libre Text Biology.

Image Courtesy:

1. “Gel electrophoresis insert comb” By Cirosantilli2 – Own work (CC BY-SA 4.0) via Commons Wikimedia

About the Author: Hasini A

Hasini is a graduate of Applied Science with a strong background in forestry, environmental science, chemistry, and management science. She is an amateur photographer with a keen interest in exploring the wonders of nature and science.

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