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Electrophoresis is a technique that separates molecules in their liquid state, based on their ability to move in an electric field. The various forms and types of electrophoresis have become the leading methods of the analysis of biomolecules in biochemistry and molecular biology, including genetic materials such as DNA or RNA, proteins, and polysaccharides.
Principle of Electrophoresis
Electrophoresis is based on the phenomenon that most biomolecules exist as electrically-charged particles, possessing ionizable functional groups. Biomolecules in a solution at a given pH will exist as either positively or negatively charged ions.
When subjected to an electric field, ionized biomolecules 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 (Andrews, 1986, as cited in Westermeier, et al., 2005, p. 3).
Theories related to electrophoresis
Electrophoretic separation occurs when an electric field is applied between two electrodes, cathode, and anode, which are submerged in a buffer solution. The following equations describe the phenomena taking place during electrophoresis, i.e. the factors affecting the electrophoretic separation (Westermeier, et al., 2005; Walker, 2010).
a. The electrophoresis setting drives the speed and direction of the particles to be separated
When an electric field is applied, voltage (V) or electric potential difference occurs. It represents the difference in the work required to move a unit of a charged particle at a certain distance (d) without producing an acceleration. The relationship between the applied electric field (E), the voltage (V), and the distance (d) in electrophoretic separation are expressed as:
For a particle possessing a total charge of q coulomb, the force (F) that pushes a charged particle is proportional to the net charge of that particle in that particular electric field:
During electrophoresis, the velocity (v) of a charged particle, at which it moves in a particular direction in an electric field, is expressed as:
where f is a frictional coefficient that is dependent on the shape and size of the particle being separated during electrophoresis and on the pore size of the medium and the velocity of the buffer solution used in electrophoresis.
The electrical mobility (µ), which is the ability of a charged particle to move in response to the applied electric field (E), can be expressed with the velocity (v) of the charged particles as follows:
In Equation (1)-(4), the electrical mobility (µ) of a charged particle is proportional to its velocity (v) and the net charge of the particle (q) but is inversely proportional to the frictional coefficient (f). Based on these equations, it is indicated that in any given electrophoresis, particles possessing different sizes and charges will mobilize at a different speed and direction, respectively, given that the applied electric field is uniform.
The inverse proportional relationship between electrical mobility (µ) and the frictional coefficient (f) also stipulates that smaller particles will migrate at a faster pace than larger particles. Thus, each particle will be separated based on its net charge, size, and shape.
b. Heat is generated during electrophoresis
Apart from the charged particle of interest, the ions in a solution buffer of the electrophoresis will also be ionized and charged once the electric field is applied. These ions serve as conductors that transport the electric current (I) between the cathode and anode. The relationship between the voltage (V) and the current (I) is described in Ohm’s law as follows:
where R is the electrical resistance that is determined by the components used in the electrophoresis setting such as the type of solution buffer being used and its total volume.
When the current (I) meets the resistance (R), electrical energy can be converted into thermal energy, termed Joule heating, also known as resistance of Ohmic heating. This phenomenon can be expressed as follows:
where P is the energy per unit time converted from electrical to thermal energy.
Based on Equations (1) and (5), the distance (d) of the electrophoretic separation can be decreased if the voltage (V) is increased, which will also lead to the increased current (I) and resistance (R). Nevertheless, as stated by Equation (6), the increase in either the current (I) or resistance (R) will result in power generation (P) that can be converted from electrical to thermal energy.
Excessive heat generation during electrophoresis can result in convection currents or transfer of heat to samples, evaporation of solution buffer, variation in the pH of the solution buffer, and thermal instability. These may lead to the mixing of samples, changes in electrophoresis setting, and damage to heat-sensitive samples.
One popular countermeasure to heat generation is to use a stabilized power supply that can provide constant voltage or current. Nevertheless, according to Ohm’s law in Equation (5) and (6), applications of constant voltage or current cannot eliminate heat generation. Therefore, the ideal electrophoresis setting should be a compromise between power, i.e. voltage and current settings, and separation time (Walker, 2010).
Forms of Electrophoresis
Based on the type of buffer solution and its effect on the mobility of the charged particles, electrophoresis can be broadly divided into four forms as follows (Jorgenson, 1986):
1. Moving boundary electrophoresis
Moving boundary electrophoresis is considered the original form of electrophoresis. Samples for separation are performed in free solution, in tubes or capillary tubes, under constant pH value throughout the separation process.
A major advantage of electrophoresis in free solution is the ability to measure the mobilization of separating particles without other intervening factors unrelated to the separating particles. Nonetheless, this electrophoresis format is vulnerable to convection current and the resolution of separation is low due to the mixing of samples in the solution buffer which can result in the overlapping of components or particles that possess similar characteristics (Jorgenson, 1986).
2. Zone electrophoresis (ZE)
Zone electrophoresis is similar to moving boundary electrophoresis in that electrophoretic separation takes place in a homogenous buffer system (Westermeier, et al., 2005). This format often makes use of a support medium or a matrix to subdue convection current and prevent uncontrolled sample diffusion. The matrix, in most cases, also provides an additional sieving effect that exerts an influence on electrophoretic separation, as shown in Equation (3) (Jorgenson, 1986; Walker, 2010).
Samples for ZE-separation are surrounded by an electrophoretic solution buffer and separated in the matrix for a definite separation time. When the electric current is applied, the samples move at a different velocity, as determined by their mass and charge (q). Once the separation process is terminated, components of the sample possessing similar characteristics are isolated into a distinct zone (Becker, 1973).
Gel electrophoresis is an example of ZE that uses a polymer-sieving matrix as a support media. The technique is widely used in biochemistry and molecular biology research and routine work due to its simplicity and versatility (Westermeier, 2005). Due to the introduction of a support medium, ZE is neither suitable for the analysis of the mobility of charged particles of interest nor the determination of the isoelectric point (pI) of peptides or proteins.
3. Isotacho-electrophoresis, or isotachophoresis (ITP)
Isotachophoresis or ITP is a form of electrophoresis, in which all ions migrate at equal velocity (v). In ITP, the samples are placed between two non-homogeneous solution buffers, composed of a leading electrolyte at one electrode, and a terminating electrolyte at the other end (Becker, 1973). Both electrolytes possess the same charge species as that of the particle of interest in the sample. When an electric current is applied, the leading electrolyte will have the highest mobility, followed by the charged particles in the sample and the terminating electrolytes, respectively.
As ITP continues, charged particles in the sample will be displaced, based on their electrical mobility (µ) and concentration, in order of decreasing mobility, resulting in a continuous region of charged particles with similar characteristics, sandwiched by regions where the leading and terminating ions are occupied (Becker, 1973; Jorgenson, 1986).
The information from isotachophoresis is conveyed as graphs of electric field strength over time, which represents the identity of the charged particle, and the length of each region represents the concentration of the charged particle (Jorgenson, 1986).
Figure 1: Diagram representing isotachophoresis separation.
The ions of the leading electrolyte (LE) have the highest mobility, followed by particles B, A, and C of the sample, respectively. Ions in the terminating electrolyte (TE) have the lowest mobility. In ITP, the identity of the charged particles is reflected in the voltage or electric field strength, depicted in the y-axis. The concentration of each particle is reflected in the duration of time it is detected (L). In this figure, Particle A has the highest concentration in the sample, followed by B and C, respectively.
One major disadvantage of this format is that only one charged species can be determined in one setting, and another round of ITP will be necessary to obtain information on charged particles of the other species.
4. Isoelectric focusing (IEF)
Isoelectric focusing or IEF is electrophoresis that is performed in a pH gradient, which runs from low to high – from the anode to the cathode. IEF is only applicable to amphoteric molecules because they can donate and receive protons, acting as acid and base. Examples of amphoteric biomolecules are peptides and proteins, which possess the amine and carboxylic acid groups (Walker, 2010).
Once the pH gradient is established, and an electric current is applied, an amphoteric sample will migrate towards either the anode or the cathode, depending on the net charge of the sample. At the isoelectric point (pI), where the net charge of the sample is zero, the velocity (v) and the electrical mobility (µ) of the amphoteric molecule become zero, stopping the migration (Jorgenson, 1986).
All four formats of electrophoresis can be performed in both one- and two-dimensions (2D). Two-dimensional electrophoresis is performed by conducting the first electrophoresis, followed by the second electrophoretic separation in a direction perpendicular to the first dimension. 2D-electrophoresis can offer more information and resolution, which is particularly useful for clinical or field samples, which often requires intensive analysis and characterization but is given only in a limited amount (Jellum & Thorsurd, 1982; Xu, 2008).
Types of Electrophoresis
The different forms of electrophoresis have been modified into several types of electrophoresis, which are used to separate various types of biomolecules, analyze their characteristics, and study their interaction with a molecule of interest. The following are selected electrophoresis methods, based on different formats of electrophoresis.
1. Gel Electrophoresis
Gel electrophoresis is a form of ZE that uses gel, a non-fluid cross-linked polymer network, as a support medium to maintain stable pH value in the solution buffer, acting as an anti-convective stabilizer. It also serves as a separation matrix, due to its porous nature that filters large particles and hinders smaller ones during electrophoretic separation (Jorgenson, 1986).
The gel is cast into strips or slabs with slots or sample wells. Once it is completely polymerized, the gel is submerged in an electrophoresis solution buffer, and samples are loaded into each well before the electric current is applied to initiate electrophoretic separation (Walker, 2010). At the end of the gel electrophoresis, components in the samples will be separated based on their mass (Westermeier, 2005).
As mentioned earlier, gel electrophoresis is one of the most used types of electrophoresis in research and routine diagnosis due to its ease-of-use and their versatility. It can be adapted to separate a variety of biomolecules by changing the type of polymers used to cast the gel and by adjusting the composition of the polymer, altering the pore size of the gel (Xu, 2008; Chung, et al., 2014).
- Polyacrylamide is a clear and transparent gel made from the co-polymerization of acrylamide monomers in the presence of a crosslinking agent, N, N-methylene-bisacrylamide, commonly referred to as ‘bis-acrylamide’. The polymerization reaction is catalyzed by ammonium persulphate (APS) and N, N, N’, N’-tetramethylene diamine (TEMED). The pore size of polyacrylamide gels is determined by the concentration of acrylamide, which must be in proportion with its crosslinking agent. Generally, a low percentage of acrylamide gel (3%-15%) is used to separate DNA and proteins. A higher percentage of acrylamide gel (10%-20%) is commonly used in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE), in which proteins are separated in denatured conditions, according to their size (Walker, 2010; Chung, et al., 2014).
- Agarose, a natural linear polysaccharide made of galactose and 3, 6-anhydrogalactose chains, extracted from agar isolated from red seaweeds (Westermeier, et al., 2005). Agarose, like agar, is stored as a dry powder. To cast agarose gel, the agarose powder is dissolved in a relevant solution buffer, heated, and allowed to cool to room temperature. Similar to polyacrylamide gel, the concentration of the agarose in the solution buffer determines the pore size of the gel. Agarose gel is commonly used at 0.8% (w/v) to 5% (w/v) to separate DNA and RNA molecules (Walker, 2010). Agarose can be used in combination with SDS to separate high-molecular-weight proteins, which can be problematic when separated using SDS-PAGE (Greaser et al., 2012).
2. Pulsed-field Gel Electrophoresis (PFGE)
Pulsed-field Gel Electrophoresis (PFGE) is a variation of gel electrophoresis, in which two electrical fields are periodically applied, in rotation, to the gel electrophoresis at different angles. This type of electrophoresis is specifically designed for the separation of chromosomes, which are high-molecular-weight DNA molecules of over 20 kilobases.
Unlike smaller DNA molecules, high-molecular-weight DNA molecules are in a compressed conformation, causing them to move in a size-independent manner. During the application of the first electric field, the coiled DNA molecules are stretched and will move through the gel. The termination and the alteration in the direction of the electric field, however, force these large DNA molecules to reorient themselves before migration can resume.
Larger high-molecular-weight molecules will be slower in the process of reorientation and migration renewal, compared to smaller high-molecular-weight molecules due to their longer viscoelastic relaxation time. Repeated application of two alternating electric fields from different angles will eventually reveal further migration distance for smaller high-molecular-weight DNA molecules and shorter migration distance for larger high-molecular-weight DNA (Westermeier, 2005; Westermeier et al., 2005; Walker, 2010).
3. Capillary Electrophoresis (CE)
Capillary electrophoresis, also known as High-Performance Capillary Electrophoresis (HPCE), is a type of electrophoresis performed in a narrow capillary immersed in an electrolyte buffer. It is the only type of electrophoresis capable of performing all four types of electrophoreses (Heiger, 2000). The capillary is typically 20-30 centimeters long and possesses a 25-75 micrometer inner diameter.
Electrophoretic separation is initiated when a sample is injected into the capillary, either by high voltage or by pressure, and high electric fields are applied across the capillary (Heiger, 2000; Westermeier, et al, 2005). Components in the sample are separated along the length of the capillary, based on the format of electrophoresis performed. Towards the other end of the capillary, separated components are detected at the detector, where the time of detection or retention time is automatically recorded.
Because CE is performed in a narrow capillary, only a very small sample volume is required for the separation. Another major advantage of CE is the automated instrumentation system, enabling high-throughput analysis (Heiger, 2000).
Immunoelectrophoresis is a type of electrophoresis that separates antigens, including proteins and peptides, based on their reaction and specificity to antibodies, or immunoglobulins (Ig). The binding of antigen to its corresponding antibody at a specific antigen/antibody ratio, or the equivalent point, will result in the precipitation of antigen-antibody complex. Thus, antigens in a sample of interest can be separated based on their ability to bind to a given antibody (Nowotny, 1979).
Contemporary settings for immunoelectrophoresis are based on modifications of zone electrophoresis and gel electrophoresis. Immunoelectrophoresis can be performed in a one-dimensional or two-dimensional mode (Westermeier et al., 2005; Walker, 2010).
5. Affinity Electrophoresis
Affinity Electrophoresis is a type of electrophoresis that separates a biomolecule that interacts with or binds to another molecule for which it has an affinity. It makes use of the phenomenon that the electrical mobility (µ) changes when a biomolecule, including nucleic acids, proteins, peptides, and polysaccharides, binds to another molecule, and this change in the electrical mobility will be reflected in the electrophoretic pattern.
Thus, biomolecules of interest in a sample can be singled out based on their affinity to another molecule—whether or not the biomolecules of interest tend to bind to another molecule more than the other unwanted biomolecule. Contemporary settings for affinity electrophoresis are based on either gel electrophoresis or capillary electrophoresis (Kinoshita et al., 2015).
All types of electrophoresis separate charged particles while they are submerged in a solution buffer. All forms of electrophoresis require a power supply and an electrophoresis unit, commonly referred to as an electrophoresis chamber. The power supply provides the electric current to the chamber that propels the electrophoretic separation. The chamber is composed of two opposite electrodes, cathode and anode, and of a buffer solution reservoir, in which the samples and the separation thereof take place (Walker, 2010).
The use of a power supply and an electrophoresis chamber is also required by all types of electrophoresis. However, each format necessitates specific equipment for the setting up of the separation process. For example, caster trays, glass plates, and combs are prerequisites for gel electrophoresis. Capillary electrophoresis requires an internal cooling system that effectively suppresses excessive heating and provides a thermally stable condition during electrophoresis.
Figure 2: Diagrams of electrophoresis unit of (A) gel electrophoresis and (B) capillary electrophoresis
Detecting electrophoretic separation
When electrophoresis is completed, the resulting product in all forms and types of electrophoresis, except capillary electrophoresis, is the separation of particles or components in the sample that are oftentimes invisible to human eyes. Therefore, additional detection and analytical methods are imperative for the interpretation of results obtained from electrophoresis (Jorgenson, 1986; Walker, 2010).
Figure 3: An example of a Gel Document System, or Gel Doc equipped with a UV light source, suitable for the dye that emits ultraviolet fluorescence when stained targeted biomolecules are exposed to UV light. The Gel Doc can be connected to a computer unit, which allows imaging of stained gels. (Taken from SynGene)
In the case of gel electrophoresis, for example, the separated particles remain embedded in the gel, invisible under normal light. To visualize the pattern of migration, the gel is stained and then visualized using a Gel Documentation System, which provides the necessary light source. The gel documentation system is frequently equipped with a camera, which can image the gel visualization for quantification or downstream analysis.
There are several approaches to detect and visualize the result of electrophoresis, each with a different degree of sensitivity and complexity. Common detection methods are as follows (Jorgenson, 1986):
- Staining uses a variety of dyes that can bind to the molecules of interest. For example, Coomassie Brilliant Blue or silver staining can bind to proteins. Ethidium bromide is an example of a dye that can insert itself between the nucleobases and fluoresces strongly under short-wave ultraviolet (UV) light. This approach is a common detection method for gel electrophoresis.
- Autoradiography can be used for detection if the molecules of interest are labeled with radioisotopes. Compared to staining, autoradiography is more sensitive.
- Online detection, which more often than not, is a part of automated instrumentation. Several detection mechanisms can be employed for online detection such as detection of UV absorption, fluorescence, electric field, and conductivity. Capillary electrophoresis and isotachophoresis employ this detection approach.
Apart from autoradiography, the quantification of separated components is relative to known standards subjected to the same electrophoretic separation. In the case of autoradiography, direct quantification can be achieved using a densitometer.
Several types of electrophoresis can be incorporated with other techniques, either for research, routine analytics, or diagnostic purposes (Walker, 2010).
- Polymerase Chain Reaction (PCR) is an in vitro technique used to generate multiple copies of a specific region of DNA. The PCR-amplified products are, in most cases, separated by gel electrophoresis or capillary electrophoresis. PCR coupled with separation and detection by capillary electrophoresis has become a standard routine method in forensic analysis of unknown DNA samples.
- Sanger sequencing is a DNA sequencing method based on the incorporation of chain-terminating dideoxynucleotides. Traditionally, Sanger sequencing is resolved using polyacrylamide gel electrophoresis. Nowadays, Sanger sequencing is resolved using laser-induced capillary electrophoresis.
- Electroblotting refers to a technique for the transfer of nucleic acids (Northern blot for RNA and Southern blot for DNA) or proteins (Western blot) onto a membrane. It is commonly performed as a downstream procedure after gel electrophoresis to allow further analyses of the separated components.
- Protein profiling with mass spectrophotometry (MS) or proteomics is a technique that attempts to determine or identify proteins in a given sample based on their mass-to-charge ratio of ions. Before mass spectrophotometry, samples are separated by certain formats or types of electrophoresis, usually in a 2D model. Then the component of interest is subjected to MS for identification.
Overall, electrophoresis is a separation technique that can single out biomolecules or charged particles of interest, based on their mobility in a given electric field. The mobility can be a reflection of the characteristics of the biomolecules or particles of interest or result from their interaction with another molecule. When combined with other techniques, electrophoresis can create a powerful and all-around tool that is applicable to research, routine analysis, and diagnosis.
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