Polyacrylamide Gel: Overview & Applications

Overview

Polyacrylamide gel is a gel made by the polymerization of acrylamide monomers. It forms due to crosslinking between polyacrylamide and bis-acrylamide molecules in the presence of a catalyst TEMED (N, N, N′, N′-tetramethylethylenediamine) and a polymerizing agent ammonium persulfate (APS that act as a free radical source to carry out the polymerization reaction). 

Polyacrylamide gel is extensively utilized in molecular biology and life sciences labs to perform gel electrophoresis techniques for the separation of biological macromolecules. The technique involving acrylamide gel is known as Polyacrylamide gel electrophoresis (or PAGE). 

PAGE also uses a surfactant or denaturant, sodium dodecyl sulfate (SDS), which denatures biomolecules' parent structures for their separation in the gel matrix based on the differences in their molecular weights. This process is known as SDS-PAGE. 

Polyacrylamide gel electrophoresis has many applications ranging from measuring the molecular weight of biomolecules and protein quantitation to detecting post-translational modification and peptide mapping.

One primary advantage of using this technique is that the gel is inert. It doesn’t affect the reactions or the chemistry of cells or assays performed by researchers, providing accurate and reliable results. 

In this article, we will review how polyacrylamide gels work, the requirements of the experiment, and their applications in different industries.

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How Does Polyacrylamide Gel Work?

SDS PAGE gel is routinely used in labs for the separation of proteins based on their molecular weight. Often it’s also used to separate nucleic acids (DNA and RNA). However, the most preferred technique to separate these molecules is agarose gel electrophoresis. 

The movement of protein molecules in the gel matrix is influenced by both the size and charge of the molecules.  A denaturant (SDS) is used to denature the secondary, tertiary, or quaternary conformation of protein molecules. The denaturant binds to the polypeptide chain, imparting a negative charge. Upon applying an electric field across the matrix, the molecules start moving towards the opposite end of the gel, having a positively charged electrode or anode. 

A protein-specific stain is used to visualize the separated bands, and their molecular weight is determined by comparing them to a standard marker (present in kDa or kilodalton). 

Normally, for protein analysis, gel electrophoresis is followed by downstream applications, such as western blotting and amino acid sequencing.  

Requirements of Polyacrylamide Gel

You need a range of reagents, equipment, and tools to perform gel electrophoresis techniques in your labs. 

Reagents

• Resolving gel 12.5% (pH of 8.8): You need 12.5% acrylamide, 0.1% bisacrylamide, 50 μl 10% (v/v) ammonium persulfate, 5 μl TEMED, 375 mM Tris-HCl, per 10 ml.
• Stacking gel (pH of 6.8): Use 5% acrylamide, 0.13% bisacrylamide, 50 μl 10% (v/v) ammonium persulfate, 5 μl TEMED, and 125 mM Tris-HCl

NOTE: Both the resolving gels and stacking gels have different ionic strengths and acrylamide concentrations that form different pore sizes for the movement of proteins in the matrix. For example, the stacking gel has a bigger pore size, which allows the faster movement of molecules. However, the mobility is slow in the resolving gel. 

• 2 × protein sample buffer (pH 6.8): 4% SDS, 20% (v/v) glycerol, 160 mM Tris-HCl, 0.05% bromophenol blue. You can also use a reducing agent, such as  2-mercaptoethanol, in the solution.
• Prestained protein molecular weight markers
• Buffer Systems: Tris-glycine-SDS (TGS) running buffer, Assay Buffer, Sample Buffer, and Gel casting buffer.
• Coomassie Brilliant Blue Stain or autoradiography for proteins

Equipment & Tools

• Glass plates
• Rocking platform or shaker
• Electrophoresis chamber
• Casting frame
• Casting stand
• Teflon spacers and Combs
• Rocking platform or shaker

What Is Polyacrylamide Gel Used For?

Polyacrylamide is frequently used in analytical techniques in labs by researchers to separate proteins based on their molecular weight and charges. The separated proteins are used for further downstream applications such as peptide mapping and amino acid sequencing.

Protein Analysis

Gel electrophoresis is used in proteomics labs for a range of applications, such as determining the size of a protein, analyzing its structure, and assessing a sample’s complexity. 

Based on the parameter a protein is separated, the gel electrophoresis technique is of two types:

• 1-D gel electrophoresis: The technique is used to separate proteins only based on their molecular weight.
• 2-D gel electrophoresis: This process is used to separate proteins based on their both isoelectric point and molecular weight. It provides better resolution than the 2D gel electrophoresis approaches. 

What Industries Use Polyacrylamide Gel?

Polyacrylamide gel electrophoresis is widely used in many Life Sciences labs and industries, including molecular biology, proteomics, and biotechnology, that perform protein research for different purposes. 

Biochemistry

Biochemistry labs use PAGE techniques for a myriad of research workflows, such as:

• Study protein structure
• Study protein interactions 
• Detect and analyze specific proteins
• Purify proteins from a sample mix
• Study enzymes and their isoforms

Biotechnology

Polyacrylamide is used in biotechnology labs to perform the separation of different biomolecules, such as DNA, RNA, and proteins. The separated fragments of the biological macromolecules are used in downstream applications of research, such as western blotting, northern blotting, proteome analysis, and next-generation sequencing. 

Forensic Chemistry

In forensic labs, polyacrylamide gel electrophoresis is used in criminal investigation for DNA profiling and identification. It’s also used in paternity tests and in identifying specific proteins for drug identification purposes. 

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Polyacrylamide gel is a gel matrix formed by a mix of polyacrylamide and bis-acrylamide molecule through a free-radical reaction initiated by TEMED and a polymerizing agent, ammonium persulfate (APS). 

The gel has paramount importance in Molecular Biology and Biochemistry labs to carry out research studies on different biomolecules, such as proteins, DNA, and RNA. The results obtained can affect further downstream applications. That’s why it’s essential to use quality reagents and advanced equipment to perform these experiments.

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