RESEARCH STARTER

Gel electrophoresis

Gel electrophoresis is a widely used laboratory technique that enables the separation and analysis of biomolecules such as DNA, RNA, and proteins based on their size. This method relies on the principle that smaller molecules can move more easily through a gel matrix than larger ones when an electric field is applied. Typically, the gel is made from agarose or acrylamide, which creates a porous structure that facilitates the movement of these molecules.

In practice, samples are loaded into wells created in the gel, and an electric current is applied, causing negatively charged nucleic acids to migrate toward the positive electrode. The distance each molecule travels is proportional to its size, allowing researchers to estimate their molecular weight. Visualization of the separated molecules is achieved using specific dyes: ethidium bromide for nucleic acids and Coomassie blue for proteins, which bind to the respective molecules and enable their detection under ultraviolet light or in visible wavelengths.

Gel electrophoresis is essential for various applications in molecular biology, including genetic research, protein analysis, and diagnostics, making it a crucial tool for scientists across disciplines.

Full Article

SIGNIFICANCE: Gel electrophoresis is a laboratory technique involving the movement of charged molecules in a buffer solution when an electric field is applied to the solution. The technique allows scientists to separate DNA, RNA, and proteins according to their size. The method is widely used way to determine the molecular weight of these molecules and can be used to determine the approximate size of most DNA molecules and proteins.

Basic Theory of Electrophoresis

Biologists often need to determine the approximate size of DNA fragments, RNA, or proteins. All of these molecules are much too small to visualize using conventional methods. The size of a piece of DNA carrying a single gene can vary widely, and DNA is approximately 2 nanometers (20 angstroms) in diameter. Therefore, some indirect method of “seeing” the length of these molecules must be used. The easiest and by far most common way to do this is by gel electrophoresis. Electrophoresis is based on the theory that if molecules can be induced to move in the same direction through a tangled web of material, smaller molecules will move farther through the matrix than larger molecules. Thus, the distance a molecule moves will be related to its size, and knowing the basic chemical nature of the molecule will allow an approximation of its relative molecular weight.

As an analogy, imagine a family with two children picnicking by a thick, brushy forest. Their small dog runs into the brush, and the whole family runs in after it. The dog, being the smallest, penetrates into the center of the forest. The six-year-old can duck through many of the branches and manages to get two-thirds of the way in; the twelve-year-old makes it halfway; the mother gets tangled up and must stop after only a short distance; the father, too large to fit in anywhere, cannot enter at all. This is what happens to molecules moving through a gel: Some travel through unimpeded, others are separated into easily visualized size groups, and others cannot even enter the matrix.

The Electrophoresis Setup

The gel is typically composed of a buffer solution containing agarose or acrylamide, two polymers that easily form a gel-like material at room temperature. At first, the buffer/polymer solution is liquid and is poured into a casting chamber composed of a special tray or of two plates of glass with a narrow space between them. A piece of plastic with alternating indentations like an oversized comb is pushed into one end of the gel while it is still liquid. When the gel has solidified, the “comb” is removed, leaving small depressions in the matrix (wells) into which the DNA, RNA, or protein sample is applied. The gel is then attached to an apparatus that exposes the ends of the gel to a buffer, each chamber of which is attached to an electric power supply. The buffer allows an even application of the electric field. Electrophoresis systems increasingly use microfluidic or ‘lab-on-a-chip’ technology, which allows faster analysis, smaller sample volumes, greater automation, and portable diagnostic testing. Capillary electrophoresis is also widely used for high-resolution, automated analysis in DNA sequencing, forensic science, clinical diagnostics, and pharmaceutical quality control, often replacing traditional slab gel methods.

Since the molecules of interest are so small, matrices with a small pore size must be created. It is important to find a matrix that will properly separate the molecules being studied. The key is to find a material that creates pores large enough to let DNA or proteins enter but small enough to impede larger molecules. By using different concentrations of agarose or acrylamide, anything from very short pieces of DNA that differ only by a single nucleotide to very large DNA fragments (using specialized methods such as pulsed-field gel electrophoresis) can be separated.

Agarose is composed of long, linear chains of multiple monosaccharides (sugars). At high temperatures, 95 degrees Celsius (203 degrees Fahrenheit), the agarose will “melt” in a buffer solution. As the gel cools to around 50 degrees Celsius (122 degrees Fahrenheit), the long chains begin to wrap around each other and solidify into a gel. The concentration of agarose determines the pore size, since a larger concentration will create more of a tangle. Agarose is usually used with large DNA or RNA molecules.

Acrylamide is a small molecule with a carbon–carbon double bond and an amino group. When the reactive chemicals ammonium persulfate and TEMED are added, the carbon ends fuse together to create long chains of polyacrylamide. If this were the only reaction, the end result would be much like agarose. However, a small proportion of the acrylamide is replaced with bis-acrylamide, a two-headed version of the acrylamide molecule. This allows the formation of interconnecting branch points formed at intervals along the polymer chains, which creates a pattern more like a net than the tangled strands of agarose. This results in a narrower pore size than agarose, which allows the separation of much smaller fragments. Acrylamide is used to separate proteins and small DNA fragments and for sequencing gels in which DNA fragments differing in size by only a single nucleotide must be clearly separated.

Why Nucleic Acids and Proteins Move in a Gel

DNA and RNA will migrate in an electric field since every base has a net negative charge. This means that DNA molecules are negatively charged and will migrate toward the positive pole if placed in an electric field. In fact, since each base contributes the same charge, the amount of negative charge is directly proportional to the length of the DNA. This means that the force on DNA or RNA is proportional to its charge, which is related to its length.

The charge on different amino acids varies considerably, and the proportions of the various amino acids vary widely from protein to protein. Therefore, the charge on a protein has nothing to do with its length. To correct for this, proteins are mixed with the detergent sodium dodecyl sulfate, or SDS (the same material that gives most shampoos their suds), before being loaded onto the gel. The detergent coats the protein evenly. This has two important effects. The first is that the protein becomes denatured, and the polypeptide chain will largely exist as a long strand (rather than being compactly bunched, as it normally is). This is important because a tightly balled protein would more easily pass through the polyacrylamide matrix than a linear molecule, and proteins with the same molecular weight might appear to be different sizes. More importantly, each SDS molecule has a slight negative charge, so the even coating of the protein results in a negative charge that is directly proportional to the size of the protein.

Once the molecules have been subjected to the electric field long enough to separate them in the gel, they must be visualized. This is done by soaking the gel in a solution that contains a dye that stains the molecules. For DNA and RNA, dyes such as ethidium bromide, a molecule that has an affinity for nucleic acids and intercalates between base pairs in the DNA helix, or safer alternatives (e.g., SYBR Safe) are used. The dye, when exposed to ultraviolet light, glows orange, revealing the location of the nucleic acid in the gel. For proteins, the dye Coomassie Brilliant Blue is usually used, a stain that readily binds to proteins of most types. Artificial intelligence tools such as GelGenie, have been introduced to automatically detect and quantify gel bands, improving accuracy and reducing manual analysis.


Bibliography

“AI Takes the Tedium out of Gel Electrophoresis with Fast, Accurate Analysis.” Phys.org, 5 May 2025, phys.org/news/2025-05-ai-tedium-gel-electrophoresis-fast. Accessed 12 May. 2026.

Dunn, Michael J., editor. From Genome to Proteome: Advances in the Practice and Application of Proteomics. Wiley-VCH, 2008.

Hames, B. D., editor. Gel Electrophoresis of Proteins: A Practical Approach. 3rd ed., OUP, 1998.

Janson, Jan-Christer. Protein Purification: Principles, High Resolution Methods, and Applications. 3rd ed., Wiley, 2011.

Jollès, P., and H. Jörnvall, editors. Proteomics in Functional Genomics: Protein Structure Analysis. Springer, 2000.

Lai, Eric, and Bruce W. Birren, editors. Electrophoresis of Large DNA Molecules: Theory and Applications. Cold Spring Harbor Laboratory, 1990.

Lee, David W., et al. From X-Rays to DNA: How Engineering Drives Biology. MIT Press, 2014.

Link, Andrew J., editor. 2-D Proteome Analysis Protocols. Humana Press, 1999.

“Microfluidics – Latest research and news | Nature.” Nature, Springer Nature, 13 Oct. 2024, www.nature.com/subjects/microfluidics. Accessed 12 May 2026.

Pennington, S. R., and M. J. Dunn, editors. Proteomics: From Protein Sequence to Function. BIOS Scientific Publishers, 2001.

Rabilloud, Thierry, editor. Proteome Research: Two-Dimensional Gel Electrophoresis and Identification Methods. Springer, 2000.

Simpson, Richard J., et al., editors. Basic Methods in Protein Purification and Analysis: A Laboratory Manual. Cold Spring Harbor Laboratory Press, 2009.

Sonagra, Amit, and Sagar J. Dholariya. “Electrophoresis.” StatPearls, 5 Aug. 2022, NIH National Library of Medicine, National Center for Biotechnology Information, www.ncbi.nlm.nih.gov/books/NBK585057/. Accessed 12 May. 2026.

Westermeier, Reiner. Electrophoresis in Practice: A Guide to Methods and Applications of DNA and Protein Separations. 5th ed., Wiley-VCH, 2016.

Full Article

SIGNIFICANCE: Gel electrophoresis is a laboratory technique involving the movement of charged molecules in a buffer solution when an electric field is applied to the solution. The technique allows scientists to separate DNA, RNA, and proteins according to their size. The method is widely used way to determine the molecular weight of these molecules and can be used to determine the approximate size of most DNA molecules and proteins.

Basic Theory of Electrophoresis

Biologists often need to determine the approximate size of DNA fragments, RNA, or proteins. All of these molecules are much too small to visualize using conventional methods. The size of a piece of DNA carrying a single gene can vary widely, and DNA is approximately 2 nanometers (20 angstroms) in diameter. Therefore, some indirect method of “seeing” the length of these molecules must be used. The easiest and by far most common way to do this is by gel electrophoresis. Electrophoresis is based on the theory that if molecules can be induced to move in the same direction through a tangled web of material, smaller molecules will move farther through the matrix than larger molecules. Thus, the distance a molecule moves will be related to its size, and knowing the basic chemical nature of the molecule will allow an approximation of its relative molecular weight.

As an analogy, imagine a family with two children picnicking by a thick, brushy forest. Their small dog runs into the brush, and the whole family runs in after it. The dog, being the smallest, penetrates into the center of the forest. The six-year-old can duck through many of the branches and manages to get two-thirds of the way in; the twelve-year-old makes it halfway; the mother gets tangled up and must stop after only a short distance; the father, too large to fit in anywhere, cannot enter at all. This is what happens to molecules moving through a gel: Some travel through unimpeded, others are separated into easily visualized size groups, and others cannot even enter the matrix.

The Electrophoresis Setup

The gel is typically composed of a buffer solution containing agarose or acrylamide, two polymers that easily form a gel-like material at room temperature. At first, the buffer/polymer solution is liquid and is poured into a casting chamber composed of a special tray or of two plates of glass with a narrow space between them. A piece of plastic with alternating indentations like an oversized comb is pushed into one end of the gel while it is still liquid. When the gel has solidified, the “comb” is removed, leaving small depressions in the matrix (wells) into which the DNA, RNA, or protein sample is applied. The gel is then attached to an apparatus that exposes the ends of the gel to a buffer, each chamber of which is attached to an electric power supply. The buffer allows an even application of the electric field. Electrophoresis systems increasingly use microfluidic or ‘lab-on-a-chip’ technology, which allows faster analysis, smaller sample volumes, greater automation, and portable diagnostic testing. Capillary electrophoresis is also widely used for high-resolution, automated analysis in DNA sequencing, forensic science, clinical diagnostics, and pharmaceutical quality control, often replacing traditional slab gel methods.

Since the molecules of interest are so small, matrices with a small pore size must be created. It is important to find a matrix that will properly separate the molecules being studied. The key is to find a material that creates pores large enough to let DNA or proteins enter but small enough to impede larger molecules. By using different concentrations of agarose or acrylamide, anything from very short pieces of DNA that differ only by a single nucleotide to very large DNA fragments (using specialized methods such as pulsed-field gel electrophoresis) can be separated.

Agarose is composed of long, linear chains of multiple monosaccharides (sugars). At high temperatures, 95 degrees Celsius (203 degrees Fahrenheit), the agarose will “melt” in a buffer solution. As the gel cools to around 50 degrees Celsius (122 degrees Fahrenheit), the long chains begin to wrap around each other and solidify into a gel. The concentration of agarose determines the pore size, since a larger concentration will create more of a tangle. Agarose is usually used with large DNA or RNA molecules.

Acrylamide is a small molecule with a carbon–carbon double bond and an amino group. When the reactive chemicals ammonium persulfate and TEMED are added, the carbon ends fuse together to create long chains of polyacrylamide. If this were the only reaction, the end result would be much like agarose. However, a small proportion of the acrylamide is replaced with bis-acrylamide, a two-headed version of the acrylamide molecule. This allows the formation of interconnecting branch points formed at intervals along the polymer chains, which creates a pattern more like a net than the tangled strands of agarose. This results in a narrower pore size than agarose, which allows the separation of much smaller fragments. Acrylamide is used to separate proteins and small DNA fragments and for sequencing gels in which DNA fragments differing in size by only a single nucleotide must be clearly separated.

Why Nucleic Acids and Proteins Move in a Gel

DNA and RNA will migrate in an electric field since every base has a net negative charge. This means that DNA molecules are negatively charged and will migrate toward the positive pole if placed in an electric field. In fact, since each base contributes the same charge, the amount of negative charge is directly proportional to the length of the DNA. This means that the force on DNA or RNA is proportional to its charge, which is related to its length.

The charge on different amino acids varies considerably, and the proportions of the various amino acids vary widely from protein to protein. Therefore, the charge on a protein has nothing to do with its length. To correct for this, proteins are mixed with the detergent sodium dodecyl sulfate, or SDS (the same material that gives most shampoos their suds), before being loaded onto the gel. The detergent coats the protein evenly. This has two important effects. The first is that the protein becomes denatured, and the polypeptide chain will largely exist as a long strand (rather than being compactly bunched, as it normally is). This is important because a tightly balled protein would more easily pass through the polyacrylamide matrix than a linear molecule, and proteins with the same molecular weight might appear to be different sizes. More importantly, each SDS molecule has a slight negative charge, so the even coating of the protein results in a negative charge that is directly proportional to the size of the protein.

Once the molecules have been subjected to the electric field long enough to separate them in the gel, they must be visualized. This is done by soaking the gel in a solution that contains a dye that stains the molecules. For DNA and RNA, dyes such as ethidium bromide, a molecule that has an affinity for nucleic acids and intercalates between base pairs in the DNA helix, or safer alternatives (e.g., SYBR Safe) are used. The dye, when exposed to ultraviolet light, glows orange, revealing the location of the nucleic acid in the gel. For proteins, the dye Coomassie Brilliant Blue is usually used, a stain that readily binds to proteins of most types. Artificial intelligence tools such as GelGenie, have been introduced to automatically detect and quantify gel bands, improving accuracy and reducing manual analysis.


Bibliography

“AI Takes the Tedium out of Gel Electrophoresis with Fast, Accurate Analysis.” Phys.org, 5 May 2025, phys.org/news/2025-05-ai-tedium-gel-electrophoresis-fast. Accessed 12 May. 2026.

Dunn, Michael J., editor. From Genome to Proteome: Advances in the Practice and Application of Proteomics. Wiley-VCH, 2008.

Hames, B. D., editor. Gel Electrophoresis of Proteins: A Practical Approach. 3rd ed., OUP, 1998.

Janson, Jan-Christer. Protein Purification: Principles, High Resolution Methods, and Applications. 3rd ed., Wiley, 2011.

Jollès, P., and H. Jörnvall, editors. Proteomics in Functional Genomics: Protein Structure Analysis. Springer, 2000.

Lai, Eric, and Bruce W. Birren, editors. Electrophoresis of Large DNA Molecules: Theory and Applications. Cold Spring Harbor Laboratory, 1990.

Lee, David W., et al. From X-Rays to DNA: How Engineering Drives Biology. MIT Press, 2014.

Link, Andrew J., editor. 2-D Proteome Analysis Protocols. Humana Press, 1999.

“Microfluidics – Latest research and news | Nature.” Nature, Springer Nature, 13 Oct. 2024, www.nature.com/subjects/microfluidics. Accessed 12 May 2026.

Pennington, S. R., and M. J. Dunn, editors. Proteomics: From Protein Sequence to Function. BIOS Scientific Publishers, 2001.

Rabilloud, Thierry, editor. Proteome Research: Two-Dimensional Gel Electrophoresis and Identification Methods. Springer, 2000.

Simpson, Richard J., et al., editors. Basic Methods in Protein Purification and Analysis: A Laboratory Manual. Cold Spring Harbor Laboratory Press, 2009.

Sonagra, Amit, and Sagar J. Dholariya. “Electrophoresis.” StatPearls, 5 Aug. 2022, NIH National Library of Medicine, National Center for Biotechnology Information, www.ncbi.nlm.nih.gov/books/NBK585057/. Accessed 12 May. 2026.

Westermeier, Reiner. Electrophoresis in Practice: A Guide to Methods and Applications of DNA and Protein Separations. 5th ed., Wiley-VCH, 2016.

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