Otis Singleton
Electrophoresis is a widely used technique in biochemistry and molecular biology, with applications in medicine and genetics. The process involves using a controlled electrical current to separate biomolecules according to their size and electric charge ratio. The history of electrophoresis can be traced back to the work of Arne Tiselius in 1931, who demonstrated that charged particles could be separated based on their charge using an electrical field. Over time, the technique has evolved with the development of zone electrophoresis methods in the 1940s and 1950s, and the increasing sophistication of gel electrophoresis in the 1960s. Today, electrophoresis machines are designed with specific functions and components to facilitate the separation of charged molecules, such as DNA, RNA, and protein molecules. Building an electrophoresis machine requires careful consideration of these components and an understanding of the underlying principles of electrophoresis to ensure accurate and effective molecular separation.
| Characteristics | Values |
|---|---|
| Purpose | To separate particles, molecules, or ions by size, charge, or binding affinity |
| Basis | Analytical technique used in biochemistry and molecular biology |
| History | Developed by Arne Tiselius in 1931; new separation processes and chemical analysis techniques continue to be developed in the 21st century |
| Key Components | Buffer, which carries the current and maintains the pH of the medium; Wicks, which connect the support medium with the buffer to complete the circuit |
| Support Media | Filter paper, gels, or liquid medium |
| Applications | Used in laboratories to separate DNA, RNA, or protein molecules; also used in population genetics and molecular biology |
| Machine Example | EPS-2014 Mini Electrophoresis System, a compact design for DNA and RNA electrophoresis |
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What You'll Learn
Understanding the chemical process of electrophoresis
Electrophoresis is a technique used to separate macromolecules in a fluid or gel based on their charge, binding affinity, and size under an electric field. It is the migration of electrically charged molecules under the effect of an electric field.
The process involves placing charged macromolecules in an electric field, where they move towards the negative or positive pole based on their charge. This technique is called anaphoresis for negatively charged particles and cataphoresis for positively charged particles. Molecules with a negative charge will migrate to the anode (positive electrode) and molecules with a positive charge will migrate to the cathode (negative electrode).
The electric field is created by a power supply, and the current is carried by a buffer solution, which also helps to maintain the pH of the medium. The support medium provides the matrix in which the separation takes place. This can be a gelatinous matrix or a gel matrix, such as agarose gel, which is commonly used in biotechnology.
Electrophoresis is used in laboratories to separate and analyse biomolecules such as proteins, peptides, nucleic acids, nucleotides, RNA, and DNA. It is the basis for many biochemical methods, such as protein fingerprinting, DNA sequencing, and Southern blotting.
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Components of the electrophoresis apparatus
The electrophoresis apparatus consists of several key components that work together to separate charged molecules. Here are the components of the electrophoresis apparatus:
Gel
The gel is a critical component of the electrophoresis apparatus, typically made from agarose or polyacrylamide. It serves as the medium in which the separation of biomolecules occurs. The gel holds the samples, and the specific type of gel used depends on the application. For example, agarose gel is commonly used for DNA separation, while polyacrylamide gels are used for protein separation.
Buffer
The buffer is a solution that provides the necessary ionic environment for the electric current to flow through the gel effectively. It also plays a crucial role in maintaining a stable pH, which is essential for the stability of the samples. The buffer establishes the pH of the system and the electrical charge on the solute. Acidic and basic buffers are used to achieve the desired pH levels.
Electrodes
The electrodes are responsible for applying an electric field across the gel. Typically made of metal, they facilitate the migration of charged molecules through the gel. The electrodes are connected to the power supply, which generates the electric current required for electrophoresis.
Power Supply
The power supply is essential for generating the electric field and current required for the electrophoresis process. It provides the energy needed to drive the movement of charged particles. The power supply is connected to the electrodes, creating the conditions necessary for the separation of molecules based on their size and charge.
Sample Wells
Sample wells are small indentations or depressions created in the gel where the samples are loaded. When the electric current is applied, the charged molecules migrate from these wells through the gel, moving at different rates based on their charge and size. The sample wells are crucial for containing the samples and facilitating their separation.
Additional Components
Other components of the electrophoresis apparatus include a cover to prevent evaporation and contamination, wicks to complete the electrical circuit, and a densitometer for quantifying the separated bands by comparing their optical densities. The support medium, such as filter paper or gel, provides the matrix for separation. The specific combination of components may vary depending on the specific type of electrophoresis being performed.
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History of electrophoresis
The history of electrophoresis for molecular separation and chemical analysis began with the work of Arne Tiselius in 1931. Tiselius, with support from the Rockefeller Foundation, developed the "Tiselius apparatus" for moving-boundary electrophoresis, which was described in 1937 in the well-known paper "A New Apparatus for Electrophoretic Analysis of Colloidal Mixtures". The method spread slowly until the advent of effective zone electrophoresis methods in the 1940s and 1950s, which used filter paper or gels as supporting media.
In the 1860s, August Toepler developed methods of optical detection of moving boundaries in liquids. This method, combined with the theoretical and experimental methods for creating and analysing charged moving boundaries, would form the basis of Tiselius's moving-boundary electrophoresis method.
In 1807, Russian professors Peter Ivanovich Strakhov and Ferdinand Frederic Reuß at Moscow University observed for the first time that the application of a constant electric field caused clay particles dispersed in water to migrate. This electrokinetic phenomenon was based on Faraday's laws of electrolysis proposed in the late 18th century and other early electrochemistry.
In the 1960s, increasingly sophisticated gel electrophoresis methods made it possible to separate biological molecules based on minute physical and chemical differences, helping to drive the rise of molecular biology and biochemistry. Gel electrophoresis and related techniques became the basis for a wide range of biochemical methods, such as protein fingerprinting, Southern blot, other blotting procedures, DNA sequencing, and many more.
In 1955, Oliver Smithies introduced starch gel as an electrophoretic substrate, enabling the efficient separation of proteins. This led to the widespread application of zone electrophoresis in biochemistry. In the 1970s, the use of gel electrophoresis for the separation and analysis of nucleic acids became more prevalent with the discovery of restriction enzymes and their application in recombinant DNA technology.
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Gel electrophoresis methods
Gel electrophoresis is a method of separating charged molecules using a gelatinous matrix and a controlled electrical current. The molecules are separated according to their size and electric charge ratio.
The process of gel electrophoresis involves loading samples into wells in a gel matrix, which is placed inside a gel box. The gel box is connected to a power supply, which provides an electrical current. The voltage and duration of the current are adjusted based on the specific experiment. The current moves the molecules through the gel matrix, separating them into distinct bands or zones.
There are two main methods of gel electrophoresis: agarose/horizontal and polyacrylamide/vertical. Agarose gel electrophoresis is commonly used for separating DNA fragments of varying sizes. The agarose gel is prepared with wells for the samples, and a current is applied, causing the DNA fragments to migrate towards the positively charged anode due to their negative charge. The rate of migration depends on factors such as DNA size, agarose concentration, voltage, and the presence of ethidium bromide. After separation, the DNA bands can be visualized under UV light after staining with an appropriate dye.
Polyacrylamide gel electrophoresis, on the other hand, is typically used for separating proteins. The protein samples are loaded into wells in the polyacrylamide gel, and the electric current causes the proteins to migrate according to their charge. The proteins can then be visualized using digital imaging systems for further analysis.
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Electrophoresis in biology and medicine
Electrophoresis is a widely used laboratory technique with applications in biology and medicine. It involves the separation of charged molecules using an electric field. The technique was pioneered by Arne Tiselius in 1931, with his development of the "Tiselius apparatus" for motion-limited electrophoresis. Since then, electrophoresis has become an essential tool for the analysis of biomolecules such as proteins, peptides, nucleic acids, and nucleotides.
In biology, electrophoresis is commonly used to separate DNA, RNA, or protein molecules based on their size and electrical charge. The process involves placing samples in a gel or other porous matrix and applying an electric current. Smaller molecules move faster through the pores in the gel, allowing for separation by size. This technique is particularly useful in genomics and proteomics research, as well as in DNA fingerprinting for forensic applications and parentage confirmation. Additionally, electrophoresis plays a crucial role in the isolation and manipulation of cloned DNA fragments and the assessment of nucleic acids in cells and tissues.
In medicine, electrophoresis is used for diagnostic purposes, particularly in the identification and quantification of protein fractions to diagnose disorders related to protein synthesis or disposal. Serum, plasma, whole blood, and hemolysate are commonly used biological specimens in diagnostic laboratory setups. The effectiveness of diagnosing medical conditions through electrophoresis is enhanced when an interprofessional team is involved, including internal medicine physicians, biochemists, and laboratory experts. This collaborative approach ensures a comprehensive patient assessment and accurate interpretation of electrophoresis patterns.
The electrophoresis apparatus consists of several key components, including buffers that carry the current and maintain the pH of the medium, and wicks that connect the support medium with the buffer to complete the circuit. Overall, electrophoresis is a powerful technique that continues to find new applications in biology and medicine, with ongoing developments in separation processes and chemical analysis techniques.
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Frequently asked questions
An electro phoresis machine is used to separate particles, molecules, or ions by size, charge, or binding affinity, using a gelatinous matrix as the basis.
The key components of an electro phoresis machine include a buffer, which carries the current and maintains the pH of the medium, and wicks, which connect the support medium with the buffer to complete the circuit.
Samples are carefully prepared and charged to one end of the gel. An electric current is then applied, and the samples run to the positive end of the tray.
Some examples of electro phoresis machines include the EPS-2014 Mini Electrophoresis System and the SymphonyIEF Isoelectric Focusing, which can handle most IEF needs.
