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Applications of electrophoresis are widely seen in the fields of molecular biology and recombinant DNA technology experiments. Separation of macromolecules such as DNA, RNA and proteins from the sample is achieved by the process of electrophoresis. Due to its accuracy in separating and studying biological components, this versatile method serves as the foundation for numerous contemporary scientific and medical breakthroughs.
It works on the principle that,
| “When charged particles are placed under the influence of a strong electric field, they are attracted towards opposite charges”. |
Thus, in this method, the positively charged ions called cations move towards a negatively charged cathode. The positively charged anode attracts negatively charged ions called anions. The applications of electrophoresis are very wide and diverse which are discussed in detail in this article below:
Figure: Electrophoresis Diagram
What is Electrophoresis?
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The meaning of electrophoresis is derived from two words-“electricity and phoresis”. Thus, it is defines as a method of separation that works under the influence of electric field. In this process, the continous movement of anions and cations results into separation of charged particles. Agarose gel electrophoresis, SDS page gel electrophoresis, 2D electrophoresis, capillary electrophoresis, zonal electrophoresis are some of the important types of electrophoresis.
- The separation is done based on the ability of the solute to move through a conductive medium under an electric field.
- The application of electrophoresisi is seen in the separation of DNA and protein by various methods.
- The pre-requisites of electrophoresis are electric current, a medium, and a buffer solution.
| The motion or movement of charged particles can be calculated by using the formula: \(V = \frac{Eq}{f}\) where, V = Velocity of charged particles E= Molecule of electric field q= Net charge of the molecule f= frictional coefficient (of the molecule) |
Applications of Electrophoresis
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Electrophoresis is used in biochemical industries to separate and analyze DNA, and separate mixtures containing biomolecules like plasmids and proteins. It is also used in molecule separation based on size, affinity, and shape. Some other important applications of electrophoresis are mentioned below in detail:
1. Analysis and Fragmentation of DNA:
DNA Analysis and Fragmentation are the most common applications of electrophoresis. When an electric current is applied to the DNA strand, it breaks down into small and large fragments.
- This is because of the changing effects of electric current applied to different fragments of DNA.
- When the electric current is discontinued, agarose or acrylamide gel (medium) is used to freeze these fragments for easier analysis.
2. Protein Detection:
Immunoelectrophoresis can be used to detect the behavior of some proteins. Abnormal protein molecules can be due to kidney disease, cancer, or sclerosis. This abnormality can be detected by electrophoresis.
- Immunoglobins are antibodies detected by immunoelectrophoresis.
- Western blotting is an advanced application of electrophoresis through which specific proteins are analyzed using antibodies.
- Antibodies can be detected and analyzed so that new treatment methods can be developed to avoid disorders or allergies.
3. Testing of Antibiotics
The purity and strength of antibiotics are tested using the applications of electrophoresis. Electrophoresis is applied to a solution containing antibiotics in the form of paper, which is then soaked in antibiotics or filled with a capillary.
- By using this method, the antibiotics can be differentiated from any impurities that may be present.
- It is also used to determine the concentration of antibiotics so that accurate doses can be prescribed.
4. Testing of Vaccine
Electrophoresis is also used in the development of modern vaccines reducing the risks of diseases like measles and pertussis. A vaccine is responsible for producing certain antibodies in our body to a foreign pathogen and electrophoresis helps in the identification of these antibodies. It is also used in checking the purity and concentration of vaccines.
5. DNA Fingerprinting
A distinct banding pattern (DNA profile) is generated by cutting DNA samples using restriction enzymes and separating the resulting fragments. It is quite possible to establish paternity or motherhood using electrophoresis observations of potential parents as well as a child.
6. Clinical Significance
Abnormalities in protein levels, which cause serious diseases such as sickle cell anemia, thalassemia, etc., can be diagnosed with the use of electrophoresis in clinical settings. Additionally, immune disorders including multiple myeloma can be clinically tested through this technology.
7. Analysis of RNA
The sequence of RNA can be detected through a technique called northern blotting. Separating RNA molecules based on their size in Northern blotting is an application of electrophoresis.
8. Forensic Science
Applications of electrophoresis are also seen in the field of forensic science in matching DNA samples to source in criminal investigations. This aids in establishing biological relationships, identifying human remains, and confirming or ruling out suspects.
Factors Affecting Electrophoresis
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Several factors are responsible for affecting the applications of electrophoresis such as:
- Sample
- Electric field
- Buffer solution
- Medium
Sample
The migration rate of the molecules is affected by the charge, size, and shape of the sample. As the charge increases, the rate of migration also increases.
- The migration rate of molecules of different shapes is affected.
- Due to frictional and electrostatic forces, the rate of migration decreases for large molecules.
Electric field
The migration of molecules is affected by voltage, current, and resistance. As the voltage increases, the rate of migration also increases.
- The rate of migration of molecules increases with an increase in current.
- The resistance of electrophoresis depends on factors like gel thickness, temperature, and buffer conductivity.
Buffer Solution
A buffer solution helps stabilize the pH of the medium. As the ionic strength of the buffer increases, the amount of current carried by the buffer also increases. The amount of current carried by the sample decreases with an increase in the ionic strength, thereby decreasing the rate of migration.
Medium
The migration rate can be affected by the type of supporting medium used. An inert medium is generally used as a supporting medium that can cause adsorption, molecular sieving, and electro-endosmosis, affecting electrophoresis.
- Adsorption reduces the migration rate and the resolution of molecule separation.
- Electro-endosmosis retards the movement of anions.
Things to Remember
- The applications of electrophoresis are observed in agriculture, food, pharmaceuticals, forensic sciences, and many other fields.
- The migration and separation of ions in a solution under the influence of an electric field is called electrophoretic mobility.
- The separation of DNA fragments based on size and charge is called Gel-electrophoresis.
- The most commonly used buffer in electrophoresis is Tris-borate-EDTA.
- Sugar polymers are used as a supporting medium in the applications of electrophoresis.
Sample Questions
Ques. What is electrophoretic mobility? (2 marks)
Ans. The solute's response to the electric field is called electrophoretic mobility. When an electric field is applied, the cations (positively charged) move toward the cathode (negatively charged), and the anions (negatively charged) move toward the anode (positively charged).
Ques. Write three applications for SDS-PAGE electrophoresis. (3 marks)
Ans. SDS-PAGE electrophoresis, also called sodium dodecyl sulfate-polyacrylamide electrophoresis, has various applications in the field of molecular biology techniques. Here are some of them listed below:
- This method is used in protein separation based on their molecular weight.
- It is used to estimate the size and purity of proteins.
- SDS-PAGE electrophoresis is also used in Western blotting.
Ques. What are the functions of the gel electrophoresis technique? (2 marks)
Ans. Gel electrophoresis is one of the types of electrophoresis.
- It separates and identifies the molecules based on their size.
- Molecules like DNA, RNA, or proteins are used.
- The gel used in this technique is agarose gel, or polyacrylamide.
Ques. Why is a buffer used in electrophoresis? (2 marks)
Ans. Buffers are necessary to transfer the charge into the medium (gel).
- A buffer is a solution used in the electrophoresis technique to stabilize the pH of the medium.
- If the pH of the medium is not stable, there will be a significant change in the structure of the protein, making it difficult to separate.
Ques. What type of buffer is used in electrophoresis? (2 marks)
Ans. The following are the types of buffers used:
- Acetate
- Pyridine
- Barbiturate citrate
- Phosphate
- EDTA
- Boric acid
Ques. What is a supporting medium? Mention their types. (2 marks)
Ans. The gel or matrix where molecules are separated is called a supporting medium. The following are the commonly used medium in electrophoresis:
- Agarose gel
- Polyacrylamide gel
- Cellulose acetate gel
- Starch gel
Ques. Give the classification of electrophoresis. (3 marks)
Ans. Electrophoresis is classified as free electrophoresis and zone-electrophoresis.
- In free electrophoresis, the migration of molecules does not require a matrix or a medium.
- For example, capillary electrophoresis and immunoelectrophoresis.
- In zone electrophoresis, the migration of molecules takes place in the medium.
- For example, paper electrophoresis, gel electrophoresis, etc.
Ques. Is isoelectric focusing an electrophoresis technique? (2 marks)
Ans. Yes, isoelectric focusing is an electrophoresis technique.
- In this technique, the separation of proteins takes place at an isoelectric pH.
- It is an example of free electrophoresis, which means that the molecules require only a free-moving solution for migration.
Ques. Name the scientist who is known as the ‘father of electrophoresis’. (1 mark)
Ans. Arnes Tisellius is known as the father of electrophoresis. He invented the molecular separation technique in 1931.
Ques. Mention any two disadvantages of electrophoresis. (2 marks)
Ans. The following are the two disadvantages of electrophoresis:
- This technique has a limited sample size.
- It is a very slow process.
Ques. What happens in Western Blotting. (2 marks)
Ans. The process of separating and identifying proteins in a complex sample is called western blotting or immunoblotting. In western blotting, polyacrylamide gel electrophoresis is used to separate the proteins.
Ques. What is the concept of molecular sieving in electrophoresis? (2 marks)
Ans. Molecular sieving plays an important role in the separation of molecules based on their size. The matrix of the gel acts as a sieve, allowing only smaller molecules to pass easily.
Ques. How long does gel electrophoresis take? (1 mark)
Ans. The duration of gel electrophoresis depends on the voltage and the concentration of gel used. This process takes place at 25℃ (room temperature) for 1.5 hours.
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