What Is Electrophoresis Definition Principle Types Procedure and Uses
Because shorter strands of DNA move much faster through the gel than longer strands, the fragments are ordered in size order. An electric current is used to move molecules across a gel for separating. The gel's pores act as a filter, allowing smaller molecules to move more quickly than larger molecules. The electrophoresis conditions can be adjusted to separate molecules in a certain size range.
Let us understand what is electrophoresis and what is electrophoresis from this article.
What is Gel Electrophoresis?
Gel electrophoresis is used for separating the charged molecules, such as DNA, based on their size.
When an electric current is passed through a gel, charged molecules travel across it. An electric current passes through the gel, creating a positive charge on one end and a negative charge on the other. The charged molecules can be separated by an electric field. In gel electrophoresis DNA molecules migrate from the negative to a positively charged electrode.
Migration
The term "migration" refers to the movement of charged molecules. Molecules move in the direction of the opposite charge. As a result, a molecule with a negative charge will be attracted to the positive end (opposites attract!). When an electric current is passed over the gel, it forms a permeable matrix, similar to a sieve, through which molecules can pass.
Smaller molecules migrate faster across the gel, covering a greater distance than larger fragments, which migrate more slowly and cover a shorter distance. As a result, the molecules are separated according to their sizes.
Gel Electrophoresis and DNA
Electrophoresis allows you to distinguish between different lengths of DNA fragments.
Because DNA is negatively charged, it will migrate to the positively charged electrode when an electric current is applied to the gel.
The fragments are arranged in size order because shorter strands of DNA pass through the gel considerably faster than longer strands.
The DNA on the gel can be seen once it has been separated using dyes, fluorescent tags, or radioactive labels. On the gel, they will appear as bands.
At the same time as the samples, a DNA marker with known length fragments is usually passed through the gel.
In gel electrophoresis DNA molecules migrate from the negatively charged electrode to the positively charged electrode.
We can calculate the length of the DNA fragments in the samples by comparing the bands of the DNA samples with those of the DNA marker.
How is Gel Electrophoresis Carried Out?
Preparing the Gel
Agarose gels are commonly used to recognize DNA fragments. The amount of agarose in the gel is determined by the size of the DNA fragments we are working with. The denser the matrix, the higher the agarose content, and vice versa. Smaller DNA fragments are separated using higher agarose concentrations, whereas larger molecules require a lower agarose concentration.
To make a gel, combine agarose powder with an electrophoresis buffer and heat to a high temperature until the agarose powder has completely melted. The molten gel is then placed on a gel casting tray with a "comb" on one end to produce wells for pipetting the sample into.
The comb is removed once the gel has cooled and set (it will now be opaque rather than transparent). Pre-made gels are now widely used. The gel is then placed in an electrophoresis tank, which is subsequently filled with an electrophoresis buffer until the entire surface of the gel is covered. The electric current is carried by the buffer. The type of buffer used is determined by the size of the DNA fragments present in the sample.
The charged molecules can be separated by an electrical field through a gel that contains small pores.
Preparing the DNA for Electrophoresis
Prior to electrophoresis, a dye is added to the DNA sample to increase its viscosity, stopping it from floating out of the wells and allowing the migration of the sample through the gel to be detected.
In the first well of the gel, a DNA marker (also known as a size standard or a DNA ladder) is added. Because the marker's fragments are of a known length, they may be used to calculate the size of the fragments in the samples.
Pipette the prepared DNA samples into the remaining wells of the gel.
The lid is then placed on the electrophoresis tank, ensuring that the gel and positive and negative electrodes are aligned properly (we want the DNA to migrate across the gel to the positive end).
Separating the Fragments
The electrical current is then turned on, which causes the negatively charged DNA to flow through the gel towards the positive side.
Shorter DNA molecules move quicker than longer strands, the ability to travel further in the time the current is running.
The migration of the loading buffer dye can be used to assess how far the DNA has migrated in the gel.
The electrical current is placed on long enough for the DNA fragments to migrate far enough across the gel to be separated, but not so long that they run off the end.
The Gel Electrophoresis Diagram is represented as follows.
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A gel is contained within a buffer tank. An electrical current is conducted across the gel as the DNA samples are deposited in wells at one end. Negatively charged DNA is attracted to the positive electrode.
Visualizing the Results
The electrical current is turned off and the gel is removed from the electrophoresis tank once the DNA has migrated far enough across the gel.
The DNA is recognized by staining the gel with a fluorescent dye that binds to the DNA and exposing it to a UV transilluminator, which exposes the stained DNA as bright bands.
The colour can also be added to the gel before it is poured.
The banding pattern of the DNA marker/size standard will be visible if the gel has run standard.
The size of the DNA in your sample can then be estimated by imagining a horizontal line running across the bands of the DNA marker. The size of the DNA in the sample can then be estimated by comparing it to the marker's nearest band.
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The lengths of the DNA fragments are compared to a marker that contains known-length fragments.
Did You Know?
Did you know the process of Electrophoresis, which is useful to the students? Let us know about it.
Under the electric fields, it is used to separate charged molecules. This method is most commonly used in molecular biology to separate DNA, RNA, and proteins based on their mass and shape. When we run DNA through electrophoresis, for example, we can differentiate between DNA molecules of various shapes and sizes.
FAQs on Electrophoresis in Chemistry Principles Types and Applications
1. What is electrophoresis in chemistry?
Electrophoresis is a separation technique that uses an electric field to move and separate charged particles in a medium based on their size and charge. In electrophoresis, ions or biomolecules migrate toward the electrode of opposite charge: cations move to the cathode (–) and anions move to the anode (+). It is widely used in analytical chemistry and biochemistry to separate DNA, RNA, and proteins.
2. How does electrophoresis work?
Electrophoresis works by applying an electric potential difference across a conductive medium, causing charged particles to migrate toward opposite electrodes. The process involves:
- Sample loading into a gel or solution.
- Application of an electric field (E).
- Movement of charged molecules according to their charge-to-mass ratio.
- Separation based on differences in size, shape, and net charge.
The rate of movement is described by electrophoretic mobility (μ), which depends on the electric field strength and frictional resistance.
3. What are the types of electrophoresis?
The main types of electrophoresis are classified based on the supporting medium and application. Common types include:
- Gel electrophoresis (agarose or polyacrylamide gels).
- Paper electrophoresis.
- Capillary electrophoresis.
- SDS-PAGE (for protein separation).
- Isoelectric focusing (separation by isoelectric point).
Each type differs in resolution, speed, and analytical application.
4. What is gel electrophoresis?
Gel electrophoresis is a method of electrophoresis that separates molecules using a porous gel matrix under an electric field. The gel (such as agarose or polyacrylamide) acts as a molecular sieve:
- Smaller molecules move faster and farther.
- Larger molecules move more slowly.
It is commonly used to separate DNA fragments, RNA, and proteins in biochemical and molecular chemistry laboratories.
5. What factors affect electrophoresis?
The rate and efficiency of electrophoresis depend on several key factors. Important factors include:
- Electric field strength (voltage applied).
- Net charge of the molecule.
- Molecular size and shape.
- pH of the buffer solution.
- Temperature and viscosity of the medium.
Changes in these variables directly influence electrophoretic mobility and separation quality.
6. What is electrophoretic mobility?
Electrophoretic mobility is the velocity of a charged particle per unit electric field strength. It is mathematically expressed as:
μ = v / E
- μ = electrophoretic mobility
- v = velocity of the particle
- E = electric field strength
It depends on the particle’s charge, size, and the viscosity of the medium.
7. What is the role of buffer in electrophoresis?
The buffer in electrophoresis maintains a constant pH and provides ions to conduct electricity. Its functions include:
- Stabilizing the charge of molecules.
- Maintaining consistent electrical conductivity.
- Preventing drastic pH changes due to electrolysis at the electrodes.
Common buffers include TAE (Tris-acetate-EDTA) and TBE (Tris-borate-EDTA) in DNA electrophoresis.
8. What is SDS-PAGE in electrophoresis?
SDS-PAGE is a type of polyacrylamide gel electrophoresis that separates proteins based on molecular mass. In this method:
- Proteins are treated with sodium dodecyl sulfate (SDS), which gives them a uniform negative charge.
- The proteins migrate through a polyacrylamide gel.
- Smaller proteins move faster than larger ones.
This ensures separation primarily by size rather than native charge.
9. What is the difference between agarose gel and polyacrylamide gel electrophoresis?
The main difference is that agarose gel electrophoresis is typically used for larger DNA fragments, while polyacrylamide gel electrophoresis (PAGE) provides higher resolution for smaller molecules. Key differences include:
- Agarose gels have larger pore sizes.
- Polyacrylamide gels have smaller, more uniform pores.
- PAGE is commonly used for proteins and small DNA fragments.
PAGE generally offers greater separation precision than agarose gels.
10. What are the applications of electrophoresis?
Electrophoresis is widely used in chemistry, biochemistry, and forensic science for separating and analyzing charged molecules. Major applications include:
- DNA fingerprinting and forensic analysis.
- Protein analysis and molecular weight determination.
- Medical diagnostics (e.g., hemoglobin variants).
- Purity testing in research laboratories.
It is an essential analytical technique for molecular characterization and separation.
