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Gel electrophoresis is a type of electrophoresis that separates molecules or components based on their size or conformation in a matrix made from gel-forming substances. Since its conception, gel electrophoresis has been modified, in terms of gel types, composition, and the set-up of the system to accommodate several aspects of the characterization of biomolecules. It is nowadays regarded as one of the fundamental analytical methods in biochemistry, molecular biology, and clinical pathology.
Principles of gel electrophoresis
The original intent of using a gel as a support matrix is to overcome the limitations of earlier support matrices for zone electrophoresis. In zone electrophoresis, liquid samples are individually mixed with a loading buffer and applied onto a restricted area or zone of the support matrix that is saturated with an electrophoresis running buffer. To initiate electrophoresis, an electric field is applied and removed when the separation is completed. Components in the sample with the same characteristics are segregated into distinct bands or zones on the support matrix. Afterward, the matrix is stained for visualization and further quantitative analyses.[4,9]
Support matrix suppresses convection currents
Support matrices in zone electrophoresis act as anti-convective stabilizers that mitigate the heat generated during electrophoresis. When the electric current is met with the electrical resistance from the ionized components of the running buffer, the support matrix stabilizes the pH of the buffer surrounding the sample. This, in turn, prevents convection current induction and allows electrophoretic separation to continue without any disruption. As a result, distinctive components obtained from zone electrophoresis are disjointed and not overlapped with nearby components, unlike the result obtained from moving boundary electrophoresis.
Support matrix from gel enhances the resolution of zone electrophoresis
Despite its obvious advantages over moving boundary electrophoresis, zone electrophoresis did not become widespread until the introduction of gel as a support matrix. This is because early matrices possess certain limitations. For example, a bed of moist starch grains provides a good resolution, but the process of non-sample protein identification is time-consuming and labor-intensive. Another matrix, the filter paper, fails to provide the resolving power that is on par with moving boundary electrophoresis. It is unsuitable for the analysis of biomolecules that exist only in a small fraction of a sample, nor can it accommodate the analysis of large-volume samples due to its absorbability.[4-6]
The introduction of gel made from potato starch overcomes the shortcomings of earlier support matrices. Contrary to filter paper, boiled starch has a low absorbability once it cools and gels. Before the gel is fully set, the liquefied gel can be cast into a block or slab of various height, length, and thickness, enabling the modification of the sample application area to accommodate both small and large sample volumes. The process of non-sample protein identification is no longer necessary.
In addition to the low absorbability, gel exerts a molecular sieving influence on the molecules of the components being electrophoresed. The net charge of the molecules of the support matrix, the shape and size of the pores in-between impose additional friction onto the electrophoresed components. Larger components will be imposed to greater friction and migrate slower than smaller components and vice versa.[3,4] In other words, gel serves not only as an anti-convective support matrix but also as a separation matrix that sieves the components during electrophoresis run based on their masses, adding to the resolving power of the technique.
The gel’s lesser absorbability and their additional molecular sieving property improve the resolving power of zone electrophoresis to the extent that it is considered superior to that of moving boundary electrophoresis. Moreover, gel electrophoresis can be easily adapted to meet the desired resolving power and other objectives by fine-tuning the gel concentration and adjusting other compositions, making the technique highly versatile.
1. Starch gel electrophoresis
Starch gels are the first gel used as a support matrix for zone electrophoresis. Starch gels were prepared using soluble potato starch at about the 10-16% (w/v) concentration in the electrophoresis running buffer. The starch solution was boiled shortly and cast in a plastic tray, with slots or slits on the top for the sample application. After electrophoresis, the gel is stained with compatible dyes to visualize the pattern of separation. Nowadays, starch gels have been replaced by agarose and polyacrylamide gels, which possess less batch-to-batch variations than potato starch.
2. Agarose gel electrophoresis
Agarose is a natural linear polysaccharide isolated from red seaweed agar. It consists of alternating chains of 1,3-linked β-D-galactose and 1,4-linked 3, 6-anhydrogalactose. Agarose gel is prepared similarly as a starch gel, using a casting tray containing a comb that molds the slots or wells for each sample. The concentration of agarose determines the resolution of the electrophoresis: the higher the concentration, the greater the resolving power. Agarose gel electrophoresis is a matrix of choice for the electrophoretic separation of linear nucleic acid fragments. It is generally suitable for the separation of DNA fragments ranging from 100 base pairs to 20 kilobase pairs. Larger DNA fragments are typically separated by pulsed-field gel electrophoresis, a modified version of gel electrophoresis.
Application of Agarose gel electrophoresis
In addition to nucleic acids, agarose gel electrophoresis applies to electrophoretic separation of proteins. A low concentration of agarose gel can be used for isoelectric focusing (IEF), a form of electrophoresis that separates amphoteric molecules based on their isoelectric point (pI). Recently, an agarose gel system combined with sodium dodecyl sulfate (SDS) has been developed to separate large proteins, ranging from 200-4,000 kDa.
3. Polyacrylamide gel electrophoresis
Polyacrylamide refers to a product of polymerization of acrylamide monomers in the presence of bisacrylamide (N,N-methylene-bisacrylamide), a crosslinking agent. The reaction is catalyzed by ammonium persulphate (APS) and N,N,N’,N’-tetramethylenediamine (TEMED), and the concentration of polyacrylamide gel is dependent on a proportion of acrylamide monomer and the crosslinking agent, bisacrylamide. To cast a polyacrylamide gel, the polymerization solution is poured between a space of two vertically glass plates that are placed in parallel and sealed at the bottom, or a glass cassette. For a vertical gel system, a comb is placed on top of the gel to create sample wells where the individual sample is applied and concentrated. In a horizontal gel system, samples are typically applied onto the surface of a strip of filter paper or other materials, which are directly placed on top of the gel.[8-9]
Application of Polyacrylamide gel electrophoresis
Polyacrylamide gels are applicable for the electrophoretic separation of both nucleic acids and proteins. For nucleic acids, agarose gels are preferable due to the toxicity of non-polymerized polyacrylamide gels, and the complexity in the preparation of polyacrylamide gels. Nonetheless, polyacrylamide gels can be modified to acquire superior resolution. Denaturing polyacrylamide gel, for example, is capable of distinguishing single-stranded DNA molecules that are only one nucleotide different, allowing the application to Maxam-Gilbert and Sanger sequencing.
For proteins, polyacrylamide gels are the norm for electrophoretic separation because they are more adaptable to accommodate several aspects of protein characterization. For example, proteins can be distinguished based on the differences in their natural structure using non-denaturing, or native polyacrylamide gels. Conversely, electrophoretic separation of proteins based on their size is achievable in higher percentage (10%-20%) polyacrylamide gels with sodium dodecyl sulfate (SDS), which denatures the proteins, allowing the proteins to migrate based on the size rather than the conformation.
Modifications of gel electrophoresis
Gel electrophoresis is regarded as a highly adaptable technique and can be, first and foremost, refined by changing the type of gels, the gel concentration, and composition. Additionally, gel electrophoresis can be further modified to meet the objectives of the analysis, as follows:
1. Two-dimensional gel electrophoresis
Additional information on the samples being analyzed can be obtained by performing additional electrophoresis in the direction that is perpendicular to the first round, or second-dimensional electrophoresis. In the case of gel electrophoresis, the gel from the first round can be directly taken for the second-dimensional analysis, or it can be processed or modified beforehand. This form of modification is applicable to both agarose and polyacrylamide gel electrophoresis.
2. Buffer system: continuous and discontinuous system
While gel electrophoresis is initially performed in a homogenous buffer system as with the case of zone electrophoresis, it can be conducted in a non-homogenous or discontinuous buffer system. This is achieved by using different gel-casting and electrophoresis running buffers and by combining two gels made from different buffers. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) is the most well-known discontinuous gel electrophoresis. In SDS-PAGE, an acrylamide gel consists of a stacking gel on the upper part and a separating gel on the lower part. Stacking gels possess larger pores than separation gels and serve as a sample concentration site. Electrophoretic separation occurs in the separating gels, which possess smaller pores.
3. Gel condition: native and denaturing gels
Another modification of gel electrophoresis is the use of denaturants in the system. Denaturants or dissociating agents are added to gel-casting, sample loading, and running buffers to destabilize intramolecular bonds of nucleic acids and proteins. Denaturing conditions force nucleic acids to exist as single-stranded molecules and proteins to unfold. As a result, the mobility of these denatured molecules during electrophoresis is solely based on their size and not their conformation. In contrast to denaturing gels, nondenaturing or native gels allow for the analysis of nucleic acids and proteins based on their conformation, which can give functional information of the biomolecules under investigation.
4. Gradient gels
Gradient gels are polyacrylamide gels whose concentrations uniformly vary throughout the gel. This is achieved by pouring the higher concentrated gel to the bottom, followed by a mixture of the higher and lower concentrated gels. Once the gel is completely polymerized, the concentration at the bottom of the gel is higher than that at the top. In other words, the sieving influence of gradient gels increases as the separating components migrate from the top to the bottom of the gel. The changing concentration of gradient gels creates a zone of sharpening effect, leading to higher resolving power than a single-percentage gel of the same range and allows for the electrophoretic separation of proteins with similar masses and the determination of the molecular diameter of proteins in their native states.
Gel electrophoresis was initially conceived as another type of zone electrophoresis but has since been adapted to accommodate other types of electrophoretic separation. In addition, gel types, their condition, and composition and the electrophoresis system can be refined to meet the goals of the analysis being performed. The resolving power of the technique, its simplicity, and versatility have made the technique a staple of biochemistry and molecular biology research, as well as clinical diagnosis.
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