Electrophoresis is a simple and sensitive separation technique in clinical and research laboratories. Since its discovery, it has been an essential tool used by biologists and chemists to separate mixtures, especially proteins and nucleic acids.
Electrophoresis consists of two words; electro, meaning electricity, and phoresis, meaning movement. Thus, it implies the migration and separation of a charged particle (ions) through a solution under the influence of an electric field.
It was first demonstrated in 1807 by Ruess, who noted the migration of particles towards the anode. It has improved from the initial crude paper electrophoresis to the modern automated system. Various improved versions are available, which apply in miniaturization, precision engineering, etc. The benefit of advancement is meeting the requirement for faster and better resolution of results.
Table of Contents
Principle of Electrophoresis
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Biological molecules, like amino acids, peptides, proteins, nucleic acids, and nucleotides, possess ionizable groups. These molecules exist in solution as electrically charged species, cations (+), or anions (-) at any given pH. Thus, the electric field allows the migration of the negatively charged molecule towards the anode (a positive terminal). In contrast, the positively charged molecule migrates towards the cathode (a negative terminal).
The separation of the molecules, ions, or colloidal particles suspended in the matrix occurs due to the force of an electric field. The molecules move through a sieve-like compound based on the molecular mass and charge ratio.
It is an incomplete form of electrolysis as the electric field is removed before the molecules reach the electrode, yet the molecules separate due to electrophoretic mobilities.
Nucleic acids have negative phosphate backbones. Hence they move towards the anode in DNA electrophoresis. Ampholytes, like proteins, bear both positive and negative charges. Such compounds have negative charge in normal conditions and migrate towards the anode. At the same time, they are positively charged in acidic conditions and move towards the cathode. Hence, protein bears a negative or positive charge depending on solvent pH and isoelectric point.
Factors Affecting the Rate of Ion Mobility
The velocity of ions depends on both inherent factors and the external environment.
Inherent factors
The inherent factors that affect the velocity of ions are:
- Charge density
- Molecular weight
- The net charge of the molecule
- Size and shape of the molecule
External factors
The external factors affecting the rate of movement of ions are:
- Electrical parameters, like current, voltage, and power
- Viscosity and pore size of supporting medium
- Temperature
- The pH of the buffer
Electrophoresis Instrument
Modern electrophoresis equipment and systems vary based on its types and forms. However, all the electrophoretic system possesses two essential components:
- Power pack
Power supply drives the movement of ionic species in the medium and allows adjustment and control of either the current or the voltage.
- An electrophoresis unit
An electrophoretic system depends on its type but essentially consists of two electrodes of opposite charge (anode and cathode), connected by a conducting medium called an electrolyte. In addition, a supportive medium is present in electrophoretic systems like gel and paper electrophoresis.
- Buffer (Electrolyte)
Buffers carry applied electric current and provide appropriate pH for the process. Conducting (running) buffers like Tris borate EDTA (TBE) and Tris-acetate acid EDTA (TAE) are commonly used.
- Supportive Medium
The supportive medium is the matrix (gel), in which biomolecules are separated. It can be in the slab or capillary form. The supportive mediums used are sugar polymers like agarose gel, polyacrylamide gel, starch gel, and cellulose acetate gel. The medium runs either vertical or horizontal gel systems in gel electrophoresis. Horizontal: agarose gel electrophoresis, and vertical: SDS-PAGE. The higher the pore size, the higher the speed traveled by charged particles.
General Procedure of Electrophoresis
The electrophoresis process has three main steps; separation, detection, and quantification.
Separation
The instrument set up is according to its type. In the gel electrophoresis, gels are prepared and cast. (http://www.tntechoracle.com/) Then placed into the electrophoresis chamber. The supportive medium can be agarose gels or polyacrylamide gels. Then appropriate buffer solution is added to the system.
After the proper setup of the instrument, the sample is placed into the medium. Then the sample is run at a specific current, voltage, or power.
Detection and Quantification
Staining with a dye or autoradiography (for radioactive samples) helps in the detection of the separated components.
Quantification is done using a densitometer or by direct measurement using an optical detection system. For example, protein is fixed by precipitating in gel with acetic acid. Methanol helps prevent the diffusion of proteins from the gel during the staining process.
Forms of Electrophoresis
Based on the forms, it is of two types; zone and moving boundary electrophoresis.
In zone electrophoresis, molecules fixed to particular zones do not interact with the surroundings. Paper, cellulose acetate strips, and gel electrophoresis are examples of it.
In the moving boundary electrophoresis, charged molecules migrate in a free-moving solution without a supporting medium. E.g., Capillary electrophoresis.
Types of Electrophoresis
Based on the nature of the supporting medium, it is of the following types:
- Agarose gel electrophoresis
- Polyacrylamide gel electrophoresis
- Cellulose Acetate Electrophoresis
- Capillary Electrophoresis
Depending on the mode of technique, it has the following types:
- Paper electrophoresis
- Isoelectric focusing electrophoresis
- Two-dimensional Polyacrylamide gel electrophoresis
- Pulse field gel electrophoresis
- Zymography
- Immunoelectrophoresis
- Capillary electrophoresis
- High voltage electrophoresis
- Isotachophoresis
- Microchip electrophoresis
Uses of Electrophoresis
It is applied for routine laboratory experiments, disease diagnosis, research-oriented separations and identification. Similarly, it is used in various other fields, like forensics, agriculture, pharmaceutical, foods, etc. Some of its applications are described below:
DNA Analysis and DNA Fragmentation
Gel electrophoresis is the core technique for genetic analysis and purification of nucleic acids for further studies or disease diagnosis.
Identifying Specific protein
- The rate of movement of macromolecules in an electric field is a helpful parameter to know any changes in amino acids regarding their charge.
- Quantitative analysis of specific serum protein classes such as gamma globulins and albumins
- It helps in the identification and quantitation of hemoglobin and its subclasses.
- It also helps in the identification of monoclonal protein in either serum or urine.
- Likewise, it helps in the separation and quantitation of significant lipoprotein classes.
- Immunoelectrophoresis helps to analyze several kinds of protein’s existence and how they behave chemically in different environments.
- It is also helpful in purifying proteins for different purposes.
- Similarly, it is useful in determining the molecular weights of protein.
Coenzymes separation
It is useful in separating and quantifying coenzymes such as creatine, kinase, lactate dehydrogenase, and alkaline phosphatase coenzyme to their respective subtypes.
Analysis of chemical compounds
- It helps analyze compounds, such as water, soil, air quality or contamination, food quality, processing hygiene, and medical forensic analysis.
- It also helps in analyzing transition metals.
- Likewise, it helps to analyze organic compounds.
- Similarly, it helps in analyzing components of pesticides.
References
- Angerish A (2018). Study of Electrophoresis Techniques and its Types. Journal of Emerging Technologies and Innovative Research. 5(9): 299-304.
- Fritsch, R., & Krause, I. (2003). ELECTROPHORESIS. Encyclopedia Of Food Sciences And Nutrition, 2055-2062. https://doi.org/10.1016/b0-12-227055-x/01409-7
- Chin et al. (2013). Electrophoresis: What does a Century-Old Technology Hold for the Future of Separation Science? International Research Journal of Applied and Basic Sciences. 7(4): 213-221.
- Westermeier, R. (2005). Gel Electrophoresis. Els. https://doi.org/10.1038/npg.els.0005335
- Wilson K & Walker J (1994). Principles and Techniques of Practical Biochemistry: Electrophoretic Techniques. 4th Ed. Cambridge University Press. 4th Ed: 425-460.
