Electrophoresis is widely used in scientific research, medicine, and forensic science to identify and analyze different molecules like proteins, DNA and RNA. Let's break down how electrophoresis works, its different types, and the equipment used to perform this method.

What is Electrophoresis?

Electrophoresis is a technique used in labs to separate and analyze molecules like proteins, DNA, and RNA based on their size, charge, and other physical properties. The word "electrophoresis" comes from two Greek words: "electro," meaning electric, and "phoresis," meaning to carry or move. In this process, molecules migrate through a gel or other medium, with smaller or more highly charged molecules moving faster when an electrical charge is applied.

How Electrophoresis Works

Electrophoresis is based on the principle that charged particles will move through a medium (such as an agarose gel or liquid) when an electric current is applied. The movement of molecules depends on several factors, including:

• Charge: Molecules with a negative charge will move toward the positive electrode (anode), while molecules with a positive charge will move toward the negative electrode (cathode).
• Size: Smaller molecules can move through the medium more easily than larger ones. So, when a mixture of molecules is placed in the electric field, the smaller molecules move faster and farther than the larger ones.
• Shape: The shape of the molecules can also affect how quickly they travel. Long, complex molecules may travel more slowly than simple, compact molecules.
• Medium: The material through which the molecules move can affect the separation. A common medium is agarose or polyacrylamide gel, which acts like a filter to slow down larger molecules more than smaller ones.

The Process Step-by-Step

1. Sample Prep: A sample containing a mixture of molecules (like DNA, proteins, or RNA) is prepared. The molecules are typically mixed with a buffer solution to help maintain a stable pH and ensure good conductivity.
2. Loading the Sample: The sample is placed in small wells at one end of a gel slab.
3. Applying Electric Field: An electric current is applied, causing the charged molecules to move through the gel. The negatively charged molecules (like DNA) move towards the positive electrode, while the positively charged molecules move towards the negative electrode.
4. Separation: As the molecules travel through the gel, they are separated based on size, charge, and shape. Smaller molecules move faster and farther, while larger molecules are slowed down.
5. Staining and Visualization: After the separation, the molecules in the gel are stained with a special dye or marker. The stained molecules can then be visualized under a UV light using electrophoresis imaging equipment. The resulting visualization is what is referred to as a "ladder band" or "dna ladder band".

The NIH National Library of Medicine has a comprehensive guide covering how to perform agarose gel electrophoresis for the separation of DNA fragments.

 

(Ladder band shown above. Image courtesy: ResearchGate)

Types of Electrophoresis

There are several different types of electrophoresis, each suited to specific types of molecules and analysis goals. The most common types include:

Agarose Gel Electrophoresis

This is one of the most commonly used types of electrophoresis, especially for DNA. Agarose gel is a porous material that allows the separation of large DNA fragments. Agarose gel electrophoresis is popular in genetics and forensic laboratories because it’s simple, effective, and inexpensive. Gels are often made in labs using powder or tablet form agarose.

• Uses: DNA analysis, genetic research, forensic analysis (such as DNA fingerprinting).
• How It Works: DNA is loaded into wells in the agarose gel. The gel is placed in a tank filled with buffer solution, and an electric current is applied. The DNA fragments, being negatively charged, move toward the positive electrode. Smaller fragments travel faster and farther than larger ones.

Polymerase Chain Reaction (PCR) Electrophoresis

PCR electrophoresis is often used alongside the polymerase chain reaction (PCR) to analyze DNA fragments. After PCR amplifies specific regions of DNA, the fragments are separated by size using electrophoresis.

• Uses: To confirm the presence or absence of specific genes, disease markers, or mutations.
• How It Works: After PCR amplifies the DNA, the fragments are separated in an agarose or polyacrylamide gel to confirm the expected size of the fragments.

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

This technique is primarily used for separating proteins based on their size. SDS-PAGE is particularly important in biochemistry and molecular biology for protein analysis.

• Uses: Protein identification, characterization of proteins, determining molecular weight of proteins.
• How It Works: In SDS-PAGE, proteins are denatured with a detergent called SDS, which gives them a uniform negative charge. This way, proteins are separated purely by size, not charge. When an electric current is applied, the proteins move through the polyacrylamide gel, with smaller proteins migrating faster.

Capillary Electrophoresis

Capillary electrophoresis is used for the separation of very small volumes of liquid samples. Instead of using a gel, the sample moves through a thin capillary tube filled with an electrolyte solution.

• Uses: DNA sequencing, protein analysis, and small molecule analysis.
• How It Works: The sample is injected into a capillary tube, and an electric current is applied. The molecules in the sample separate based on their size, charge, and interactions with the capillary's inner surface.

Isoelectric Focusing (IEF)

Isoelectric focusing is used to separate proteins based on their isoelectric point (pI), which is the pH at which a protein carries no net charge.

• Uses: Protein analysis, especially for identifying different forms of the same protein (isoforms).
• How It Works: The protein sample is placed in a pH gradient in a gel, and when an electric field is applied, the proteins move until they reach the point where their net charge is zero, which is their isoelectric point.

Equipment Used in Electrophoresis

Electrophoresis requires several key pieces of equipment to perform the experiment. Let’s take a look at the most commonly used tools.

Electrophoresis Tank (Chamber)

This is the container where the gel and the buffer solution are placed. The electrophoresis tank also houses the electrodes that provide the electric field needed for the separation of molecules.

• Description: Typically made of plastic, these tanks hold the gel, buffer solution, and electrodes. They come in various sizes, depending on the scale of the experiment.

Power Supply

Electrophoresis power supplies provide the electric current that drives the molecules through the gel. The voltage is adjustable, depending on the size of the gel and the molecules being separated.

• Description: Power supplies usually have knobs or digital buttons to control the voltage and current, and they often come with safety features to prevent overloads.

 

 

Gels and Buffers

The gel is the medium that holds and separates the molecules, and the buffer solution ensures that the gel stays at the correct pH and helps conduct electricity.

• Description of Gel: Agarose gels are commonly used for DNA electrophoresis, while polyacrylamide gels are used for protein analysis.
• Description of Buffers: Buffers are essential for maintaining a consistent pH throughout the process. Common buffers include TAE or TBE for DNA electrophoresis.

Gel Casting Equipment

To make the gel, a gel casting tray and comb are used. The tray holds the liquid gel as it solidifies, and the comb is used to create wells where the sample is loaded.

• Description: The tray is usually rectangular and made of plastic. The comb is inserted into the liquid gel before it hardens to form the wells.

Staining and Imaging Tools

After the electrophoresis run, the separated molecules are stained with a special dye that binds to the molecules and makes them visible.

• Common Stains: Ethidium bromide (for DNA) or Coomassie Brilliant Blue (for proteins).
• Imaging: After staining, the gel can be visualized under UV light or a gel documentation system to capture the image of the separated bands.

Micropipettes

Micropipettes are used to precisely measure and load the samples into the gel wells. These instruments are essential for accurate handling of small liquid volumes.

• Description: Micropipettes can handle very small volumes, ranging from 1 µL (microliter) to 1000 µL (milliliter).

Applications of Electrophoresis

Genetic Research

Electrophoresis is widely used in genetic research to analyze and separate DNA, RNA, and proteins. In DNA analysis, gel electrophoresis helps visualize DNA fragments after techniques like PCR or restriction enzyme digestion, aiding in genetic fingerprinting, mutation detection, and genome mapping. Agarose gel electrophoresis is commonly used for DNA and RNA separation, while polyacrylamide gel electrophoresis (PAGE) provides higher resolution for smaller fragments. Additionally, capillary electrophoresis is used in automated DNA sequencing, including Sanger sequencing. This technique is essential for studying genetic disorders, evolutionary biology, and forensic identification.

Forensic Science

Electrophoresis is a key technique in forensic science for analyzing biological evidence, particularly DNA and proteins. Gel electrophoresis is used to separate DNA fragments obtained from crime scene samples, such as blood, hair, or saliva. In DNA fingerprinting, forensic scientists use capillary electrophoresis with STR (Short Tandem Repeat) analysis to compare suspect and crime scene DNA. This helps in criminal identification, paternity testing, and missing person investigations. Additionally, protein electrophoresis can analyze blood or other bodily fluids for forensic toxicology. The precision of electrophoresis makes it a crucial tool in forensic investigations.

Medicine

Electrophoresis is widely used in medicine for diagnosing diseases and analyzing biological molecules. Protein electrophoresis helps detect abnormalities in blood proteins, aiding in the diagnosis of conditions like multiple myeloma and immune disorders. Hemoglobin electrophoresis is used to identify hemoglobin variants, crucial for diagnosing disorders like sickle cell anemia and thalassemia. In genetic and molecular diagnostics, electrophoresis is used to analyze DNA and RNA, assisting in disease detection, genetic mutation identification, and prenatal testing. This technique is essential in clinical laboratories for diagnosing and monitoring various medical conditions.

Biotechnology

In biotechnology, electrophoresis is a fundamental technique used for analyzing and purifying biomolecules such as DNA, RNA, and proteins. Agarose gel electrophoresis is commonly used to separate and visualize nucleic acids in genetic engineering, cloning, and PCR validation. Polyacrylamide gel electrophoresis (PAGE) is essential for protein analysis, including assessing purity, molecular weight determination, and detecting post-translational modifications. Capillary electrophoresis is used in high-throughput DNA sequencing and quality control in pharmaceutical and biotech industries. This technique plays a crucial role in drug development, genetic engineering, and biomedical research.

Conclusion

Electrophoresis is widely used in modern science and medicine. By separating molecules based on their size, charge, and other properties, researchers can analyze DNA, proteins, and other biomolecules in great detail. The various types of electrophoresis, along with the specialized equipment used, make it a widely applicable technique in research and diagnostics.