What is the Difference Between Agarose and Polyacrylamide

The main difference between agarose and polyacrylamide is that agarose is used in the agarose gel electrophoresis (AGE) mainly for the separation of DNA, whereas polyacrylamide is used in the polyacrylamide gel electrophoresis (PAGE) mainly for the separation of proteins. Furthermore, agarose can separate DNA fragments of 50-20,000 bp in size while polyacrylamide has a more resolving power, separating up to 5-500 bp fragments of DNA. Moreover, agarose gels lie flat on the table with a horizontal run while polyacrylamide gels stand on the table with a vertical run. 

Agarose and polyacrylamide are the two main types of gels used to separate macromolecules, including DNA, RNA, and proteins based on their size and charge. 

Key Areas Covered 

1. What is Agarose
     – Definition, Composition, Uses
2. What is Polyacrylamide
     – Definition, Composition, Uses
3. What are the Similarities Between Agarose and Polyacrylamide
     – Outline of Common Features
4. What is the Difference Between Agarose and Polyacrylamide
     – Comparison of Key Differences

Key Terms 

Agarose, DNA, Gel Electrophoresis, Polyacrylamide, Resolving Power

What is Agarose 

Agarose is the natural polysaccharide polymer that is used in the agarose gel electrophoresis (AGE). However, it is a complex mixture of molecules and the main component of agarose is agar. Importantly, in an agarose gel, it is possible to separate large fragments of DNAs based on their size. Usually, DNA is a negatively charged molecule, which runs towards the positive electrode under an electric field. Also, pores inside the agarose gel are responsible for the separation of DNA by size.

Figure 1: Agarose Gel Electrophoresis

Furthermore, agarose gel is a 3D-matrix made up of helical agarose molecules, which aggregate into channels and pores. Still, its pore size is not uniform. Therefore, the results are difficult to reproduce. On the other hand, it is easy to handle and cast. Also, the agarose sets when the gel cools down.  

What is Polyacrylamide 

Polyacrylamide is the substance that is used in the preparation of polyacrylamide gel electrophoresis (PAGE). Basically, it is a product of crosslinking of two molecules; acrylamide and bis-acrylamide. Also, acrylamide is a product of hydration of acrylonitrile by the enzyme nitrile hydratase. In addition to these, it is in the form of a powder, which is toxic to the nervous system of humans. With the addition of water, acrylamide undergoes polymerization into polyacrylamide.

Figure 2: PAGE

Moreover, polyacrylamide gels can be in two stages; native polyacrylamide gels and denaturing gels. Generally, in the native polyacrylamide gels, the higher-order structure of the biomolecule is kept as it is. However, in a denaturing gel, the denaturing agent especially, SDS (sodium dodecyl sulfate) denatures the biomolecule, making its mobility to depend on the size. For instance, polyacrylamide gels are important for the separation of proteins as well as small nucleic acids such as oligonucleotides, tRNAs, etc. 

Similarities Between Agarose and Polyacrylamide 

• Agarose and polyacrylamide are the two main types of gels that are used for the separation of biomolecules, such as DNA, RNA, and proteins. 
• Therefore, they are important in molecular biology and biochemistry. 
• These biomolecules move between electrodes after applying the electric field, moving the charged molecules through the matrix. 
• On that account, both types of gels allow the separation of biomolecules based on their size and charge. 

Difference Between Agarose and Polyacrylamide 

Definition 

Agarose is the main constituent of agar used, especially in gels for electrophoresis. Polyacrylamide is a synthetic resin made by polymerizing acrylamide. It is a water-soluble polymer used to form a stabilized gel. 

Making 

Agarose is a complex polysaccharide derived from seaweed while acrylamide is made by the digestion of acrylonitrile by nitrile hydratase. 

Composition 

Agarose consists of many molecules, while polyacrylamide contains one large molecule. 

Gels 

Agarose gels contain long chains of interlinked sugars to form a meshwork, while the polyacrylamide gels compose chemical crosslinking of acrylamide and bis-acrylamide, producing a molecular sieve. 

Setting 

Agarose sets as it cools while polyacrylamide sets through a chemical reaction once crosslinking occurs. 

Run Configuration 

Agarose is a horizontal gel while polyacrylamide is a vertical gel. 

Properties 

The pore size of the agarose gel becomes smaller with the increasing concentration of agarose in the gel. But, the ratio of acrylamide to bis-acrylamide determines the pore size of the polyacrylamide gel. 

Typical Concentrations of the Gel 

Typical concentrations of agarose gel are around 0.5 to 2%, while the typical concentrations of polyacrylamide gel are around 6-15%. 

Separation of Biomolecules 

Agarose gels are mainly important in the separation of much larger DNA fragments such as the products of PCR while polyacrylamide gels are important for the separation of proteins as well as small nucleic acids such as oligonucleotides, miRNA, tRNAs, etc. 

Separation of DNA 

Agarose can separate DNA about 50-20,000 bp in size while polyacrylamide can separate DNA about 5-500 bp in size. 

Resolving Power 

Agarose gels have a comparatively low resolving power while polyacrylamide gels have a high resolving power. 

DNA Configuration 

Agarose gels typically separate DNA in the double-stranded form while polyacrylamide gels separate DNA in the single-stranded form. 

Pros 

Agarose gels are non-toxic and easy to handle while polyacrylamide gels give reproducible results. 

Cons 

Agarose gels do not have a uniform pore size while polyacrylamide gels contain a potent neurotoxin. 

Conclusion 

Agarose is the polysaccharide of seaweed important in the production of gels mainly to separate large fragments of DNA. Also, it is a horizontal gel with comparatively low resolving power. On the other hand, polyacrylamide is another type of gel that is mainly used in the separation of proteins. Also, it is important in the separation of small nucleic acids due to its high resolving power. In addition to that, it is made up of the crosslinking of acrylamide and bis-acrylamide through a chemical reaction. On the other hand, polyacrylamide is a vertical gel. Therefore, the main difference between agarose and polyacrylamide is the composition, setting, and resolving power. 

References:

1. Clore, Adam. “Running Agarose and Polyacrylamide Gels.” INTEGRATED DNA TECHNOLOGIES, Integrated DNA Technologies, Inc., 20 Sept. 2017. Available Here.

Image Courtesy:

1. “Electrophoresis – Moving along gel” By michael – moving along (CC BY 2.0) via Commons Wikimedia    
2. “Gelelektrophoreseapparatur” (CC BY-SA 3.0) via Commons Wikimedia