Introduction
Electrophoresis is a laboratory technique used to separate DNA, RNA, or protein molecules based on their size and electrical charge. An electric current is used to move molecules to be separated through a gel. Pores in the gel work like a sieve, allowing smaller molecules to move faster than larger molecules. The conditions used during electrophoresis can be adjusted to separate molecules in the desired size range. In the year 1807, Ferdinand Frederic Reuss was the first person to observe electrophoresis. He was from Moscow State University. Anaphoresis is the electrophoresis of negatively charged particles or anions, whereas cataphoresis is the electrophoresis of positively charged ions or cations. Electrophoresis has a wide application in separating and analyzing biomolecules, such as proteins, plasmids, RNA, DNA, and nucleic acids. To get a clear view of the different types of electrophoresis techniques, it is necessary to understand the electrophoresis principle. Electrophoresis is the process of separating the macromolecules present in a gel or fluid based on their binding affinity, size, and charge under an electric field. It was Ferdinand Fredric Reuss who observed electrophoresis for the first time in 1807. The primary classification of electrophoresis includes procedures like Anaphoresis and Cataphoresis. Anaphoresis is the electrophoresis of negatively charged elements called anions. On the other hand, Cataphoresis is the electrophoresis of cations or positively charged particles. The process has several uses in the analysis and separation of biomolecules like plasmids, proteins, nucleic acids, DNA, and RNA.
Working Principle of Electrophoresis
Charged macromolecules placed in an electric field move in the direction of the positive or negative pole. The movement ultimately depends on the charge of the macromolecules. In this context, you should know that since nucleic acid is a negatively charged particle, it tends to move in the direction of the anode. The entire electrophoresis procedure has two varieties; they are capillary electrophoresis and slab electrophoresis. Proteins, if negatively charged, will move towards the anode and the cathode if they have a positive charge. Because smaller molecules migrate faster than larger molecules, scientists can easily measure the travelled distance and make use of logarithms for determining the size of the particles.
The Principle of Electrophoresis
The reason electrophoresis works is owed to one of the fundamental equations in the physics of electromagnetism: force equals electric charge times the strength of the field at that point. This assumes the form:
F = qE,
Where F = force, q = electric charge, and E = electric field strength.
This equation implies that the higher the charge on a particle, the stronger the force that results from the application of a given electric field. This means that two particles of the same mass but different charges will move at different rates through the field. In addition, the speed at which any charged molecule moves is dependent on its charge-to-mass ratio. Together, these properties and relationships make it possible for scientists to separate the components of critical bio-molecules, such as nucleic acids, into their smaller components.
Different Types of Electrophoresis
The image depicts the types of electrophoresis.
Modern-day electrophoresis devices come with supporting media. This supporting medium is a kind of physical support that helps in separating the charged macromolecules. This physical support renders two essential functions - molecular sieving and adsorption of taken macromolecules for separation.
Some of the most commonly utilised supporting mediums include agar, agarose, starch, and polyacrylamide. Based on the availability or unavailability of the supporting medium, the electrophoresis procedure is of two categories: Capillary electrophoresis and Slab electrophoresis.
Capillary Electrophoresis (CE): Capillary electrophoresis (CE) is a family of electrokinetic separation methods performed in sub-millimeter diameter capillaries and micro and nano-fluidic channels. Very often, CE refers to capillary zone electrophoresis (CZE), but other electrophoretic techniques include capillary gel electrophoresis (CGE), capillary isoelectric focusing (CIEF), capillary isotachophoresis, and micellar electrokinetic chromatography (MEKC) In CE methods, analytes migrate through electrolyte solutions under the influence of an electric field. Analytes can be separated according to ionic mobility and/or partitioning into an alternate phase via non-covalent interactions. Additionally, analytes may be concentrated or "focused" using gradients in conductivity and pH.
Slab Electrophoresis (SE): It is a popular method for separating proteins. You can analyse many samples at a time using a 1D format. Though CE needs specific instrumentation, SE needs nothing.
These two types further can be divided into other types of electrophoresis techniques. In the capillary electrophoresis category, one can find techniques like paper electrophoresis and gel electrophoresis.
Slab electrophoresis has additional sub-categories, like zone electrophoresis, isoelectrofocusing, and immunoelectrophoresis.
We will be looking into the details of all these different types of electrophoresis below:
Gel Electrophoresis
It is one of the most preferred electrophoresis procedures in the majority of the experimental environments. There are three essential varieties of gel electrophoresis. They are starch gel electrophoresis, polyacrylamide gel electrophoresis, and agarose gel electrophoresis. In the starch gel electrophoresis procedure, potato starch granules are used in the form of a supporting medium.
Things are different in the agarose gel electrophoresis technique. Here, a wholly purified polysaccharide in large molecular mass is used as the support media. Polyacrylamide gel electrophoresis is one of the most common techniques due to its high stability. Also, it works on a large assortment of molecular concentrations.
Paper Electrophoresis
The technique is quite simple. The sample intended to be separated is applied to a strip of paper moisturised using a kind of buffer solution. There are separate tanks of this buffer solution, and each end of the paper is dipped in these tanks. Also, there is a different cathode or anode. Next, an electric current is applied. It forces the sample to move in the direction of the electrode with the opposite polarity. Once the procedure is completed, the paper is dried and then viewed using a sound quality detection system.
Immunoelectrophoresis
The process is a blend of electrophoresis and immune-diffusion. The process involves placing antigen mixture into well cuts in gel without antibodies and separating their components through electrophoresis.
Zone Electrophoresis
ZE or Zone electrophoresis is the process for the analysis of nucleic acids, biopolymers, and proteins. This electrophoretic separation procedure involves transporting different species in a buffer system under an electric current. Because of differences in mobilities, these species will separate into well-resolved and varied peaks.
Isoelectrofocusing
IEF or Iso Electrofocusing is the process of separating charged macromolecules, generally peptides or proteins. The separation depends on their isoelectric point or the pH at which a particular molecule does not have any charge. This process works mainly because the macromolecules in the pH gradient tend to move towards their pI in an electric field. IPG strips consisting of acrylamide gel with wide pores for preventing sieving effects are used for the procedure.
FAQs on Types of Electrophoresis
1. How Does the Electrophoresis Procedure Work?
During the electrophoresis procedure, an electric current is applied to macromolecules. Since the macromolecules are already electrically charged, it forces them to act upon the electric field. Molecules with higher charges will have more force applied on the part of the electronic field.
Therefore, with the support medium in place, the macromolecule will be moving with its weight. A few good examples of the uses of the process of electrophoresis are RNA and DNA analyses. Protein electrophoresis is a medical process used for analyzing and separating the molecules that are d found in fluid samples which are mainly urine and blood samples.
2. What Happens in Case the Gel Electrophoresis Technique Takes too Long?
The process of gel electrophoresis is suitable for separating proteins or DNA based on their size. This process can even be used for separating mixtures, isolating them and for identifying molecules. Now, coming to the answer to the question of what happens in case the gel electrophoresis technique takes too long to complete? The probable answer to this question is that the sample used for the process might run out of the gel bottom.
3. What is the theory of Electrophoresis?
Electrophoretic separations are based upon the fact that the electrical force (F) on a charged particle (ion) in an electrical field (E) is proportional to the charge of the particle (q), or F = qE (Eq 1). The migration of the charged particle in the electric field, called the electrophoretic mobility (μ), is defined as μ = v/E = q/f (Eq 2), where v is the velocity of the charged particle and f is a complex term called the frictional coefficient. The frictional coefficient relates to the size and shape of the particle. From equation (2), it can be seen that electrophoretic mobility decreases for larger particles and increases with a higher charge.
4. What is the basic principle of Electrophoresis?
Electrophoresis is based on the phenomenon that most biomolecules exist as electrically-charged particles, possessing ionizable functional groups. Bio-molecules in a solution at a given pH will exist as either positively or negatively charged ions. When subjected to an electric field, ionised bio-molecules will migrate at a different pace, depending on the mass and the net charge of each particle in the solution — negatively-charged particles, anions, will migrate towards a positively charged electrode, or cathode, and cations, or positively-charged particles, will be pulled towards a negatively-charged electrode called the anode. The differences in the speed and the direction of each charged particle will result in a migration pattern that is unique to its individual property, leading to the isolation of components of the biomolecules that possess similar characteristics.
5. What is the principle of protein electrophoresis?
The separation of proteins by electrophoresis is based on the fact that charged molecules usually migrate through a matrix/medium upon application of an electrical field. The rate at which proteins move in an electric field is determined by several factors of the electrophoretic system and the nature of proteins themselves. Some factors to mention are the strength of the electric field, the temperature of the system, pH of the ions, the concentration of the buffer, etc. Proteins vary in size and shape and have the charges determined by the dissociation contents of their amino acids. Smaller proteins usually migrate faster, and larger proteins take a longer time. This physical property of proteins is exploited for its separation by employing the electrophoretic technique.
6. What is the difference between electrophoresis and immunoelectrophoresis?
The major difference is that electrophoresis is the migration of electrically charged molecules through a medium under the influence of an electric field, while immunoelectrophoresis is a technique using a combination of protein electrophoresis and an antigen-antibody interaction to separate mixtures of proteins and identify them.
7. How does Electrophoresis work?
The electrophoresis process is a method that uses an electric field for separating the charged particles like DNA and many other macromolecules, such as RNA and proteins, depending on their size and charge. This is carried out by using an electrode pair that generates an electrode potential E. This helps in the migration of the charged particles towards their respective poles. The electrophoresis process is also useful in many applications, such as separating the nucleic acids and proteins, separating the DNA fragments, visualising the molecular makers, and checking the quality and quantity of the genomic DNA.