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Polyacrylamide gel electrophoresis is one of the first forms of gel electrophoresis. Polyacrylamide is a synthetic polymer that is considered the most all-around separation matrix to date due to its transparency, electrical neutrality, and adjustable pore size. The technique can be applied to the analysis of proteins and nucleic acids such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
Polyacrylamide As A Matrix For Gel Electrophoresis
Polyacrylamide gel electrophoresis (PAGE) is a form of gel electrophoresis that uses polyacrylamide gel as a support or a separation matrix. Similar to other forms of electrophoresis, polyacrylamide gels in PAGE stabilize the pH in the system and prevent convection current that is induced during electrophoretic separation. The shape and size of the space in-between polyacrylamide molecules also exert the molecular sieving influence, which imposes a frictional force on the components in the sample. The pore size can be adjusted to meet the expected size range of the resolved components. The gel is submerged in the electrophoresis running buffer, and samples in the liquid phase are mixed with a sample loading buffer containing a tracking dye, and applied to the sample application well on the top of the gel.
Electrophoretic separation begins when an electric field is applied to the unit. The components in the sample are gradually separated into distinct bands based on their characteristics, and the migration continues until the electric field is removed. Larger components will migrate at a slower rate than smaller ones because they are inflicted with greater frictional force. The result of the separation can be visualized by staining the gel with dyes that are compatible to the gels and the samples.
Polyacrylamide Gels Are Cast By Free-Radical Polymerization
Polyacrylamide is a synthetic polymer that results from the polymerization of acrylamide and N,N’-methylenebisacrylamide, also referred to as ‘bis-acrylamide’. Both acrylamide and bis-acrylamide in their monomeric forms are extremely toxic, partly due to their aqueous forms, which make them easily absorbed by the skin. The polymerization is catalyzed by ammonium persulfate or potassium persulfate and by N,N,N’,N’-tetramethylethylenediamine (TEMED). The addition of persulfate and TEMED accelerates the polymerization by increasing the formation of persulfate free radicals that convert acrylamide monomers to free radicals. Acrylamide free radicals react with unactivated monomers, which in turn, initiate the polymerization of acrylamide monomers in a head-to-tail fashion. Bis-acrylamide is cross-linked between acrylamide molecules in the polymerizing chain, forming a transparent, charge-neutral and inert polyacrylamide gel.
Since polymerization involves the generation of free-radical, the presence of oxygen can interfere with the gelation process because it can absorb the generated free radicals. To circumvent such interference, polyacrylamide solutions are usually placed under a vacuum condition to degass, which will remove air dissolved in the solutions before the polymerization is initiated. Another popular approach in reducing exposure to oxygen is to let the polymerization occurs in a vertically-placed sealed cylinder tube or the space between two glass plates to create a slab gel.
Alternatively, ammonium persulfate and TEMED can be replaced with riboflavin or riboflavin-5’-phosphate to catalyze the polymerization, in a process called photopolymerization. In the presence of light and oxygen, riboflavin degrades and generates free radicals that react with acrylamide monomers. In the case of photopolymerization, the gelation process usually takes longer than the typical polyacrylamide polymerization.
The Ratio Of Acrylamide And Its Crosslinker Determines The Pore Size Of Acrylamide Gels
Polyacrylamide gels’ pore size is determined by the concentration of acrylamide and its cross-linking agent. The ratio between acrylamide and its cross-linker, bis-acrylamide is usually adjusted to obtain the desired sieving effect on the components being separated. The relationship between the pore size of acrylamide gels, the concentration of total acrylamide and bis-acrylamide can be expressed as a percentage of total acrylamide concentration (T) and the degree of cross-linking (C) as follows:
where T is the total acrylamide concentration,
C is the degree of cross-linking
a is the mass of acrylamide in g,
b is the mass of bis-acrylamide in g, and
V is the total volume of polyacrylamide solution in mL.
As a rule, the pore size increases as the T value increases and C remains constant. When T remains constant, however, the pore size is largest at high and low C values. The C-value of polyacrylamide gels is typically 3% for the electrophoretic separation of proteins and 5% for that of nucleic acids, which are translated to 29:1 acrylamide/bis-acrylamide and 19:1 acrylamide/bis-acrylamide, respectively. Higher percentage of C-value causes the gels to be brittle, opaque and relatively hydrophobic, making them difficult to handle and of little use in electrophoretic separation.
In general, PAGE is performed using gels containing 3-30% acrylamide. Gels with lower acrylamide content (3-7.5%) are often used in the electrophoretic separation of nucleic acids, while those made with higher acrylamide content (8-30%) are used in the electrophoresis of proteins.
The Property Of Polyacrylamide Gels Can Be Altered By Changes In Their Compositions And Polymerization Reaction
Other than the pore size of the gel, PAGE can be modified by changing the setup and adjusting the gel composition to accommodate the objectives of the analysis. Since a polymerization reaction must be initiated for polyacrylamide gels, all necessary components must be present in a suitable proportion and condition. This is to ensure that polymerization can occur without any inhibition and that the gels possess the desired characteristics. The following are examples of alterations that could affect the property of acrylamide gels and the subsequent PAGE:
1. Gel additives
Similar to agarose gel electrophoresis, PAGE can be modified to separate components in their denatured state by adding additives to the gels. Denaturants such as sodium dodecyl sulfate (SDS) and Triton X-100 can be added to polyacrylamide solution without affecting the gel’s property besides its denaturing state. Other additives such as urea and formamide, however, not only denature the molecules of the components being separated but also disrupt the hydrogen bonds of the acrylamide monomers, resulting in the change of the gel’s molecular sieving influence. The pore size of urea or formamide-containing polyacrylamide gels is generally smaller than the usual polyacrylamide gels.
2. Alternative crosslinkers and monomers
Other than bis-acrylamide, other cross-linking agents can be replaced to alter the property of the polymerized acrylamide gels. N,N′-Bis(acryloyl)cystamine (BAC), N’,N’-(1,2-dihydroxyethelyne)bisacrylamide (DHEBA) and ethylene diacrylate (EDA), for example, can substitute bis-acrylamide to cast reversible gels, which can be solubilized after electrophoresis to recover the separated components. Another example of alternative crosslinker is piperazine diacrylamide (PDA), which enhances the resolution of the polyacrylamide, while reducing the background of the gel that occurs from silver staining.
Similar to bis-acrylamide, several monomer species have been synthesized to replace acrylamide. Acryloylaminopropanol (APP) is an example of a safer alternative to acrylamide, which is known to be carcinogenic and highly toxic to the nervous system.
3. The concentration of the catalysts
The two catalysts in the polyacrylamide solution, ammonium persulfate and TEMED, initiate the polymerization of acrylamide monomers. Their absence or insufficient concentration extends the duration of the polymerization, which, if too long, could inadvertently allow oxygen to dissolve in the polyacrylamide solution, leading to non-uniform pore size and mechanically weak gel. In contrast, too high a concentration of either catalyst results in shorter polyacrylamide chains, lesser gel transparency and gel elasticity. In some cases, the excess catalysts can react with the components in the samples, altering the characteristics of the components being electrophoresed.
4. Polymerization temperature and time
As with all chemical reactions, the polyacrylamide gel is sensitive to the temperature at which the polymerization takes place and the length of time it occurs. The temperature has a direct effect on the rate of polymerization, which also affects the quality of the gel. Higher temperatures can, on one hand, accelerate the polymerization reaction; on the other, it can result in the formation of shorter polyacrylamide chains that lead to brittle and opaque gels. On the contrary, lower polymerization temperature delays the gelation, and the resulting gel will be opaque, inelastic and porous. Typically, a temperature of 23-25°C is considered optimum for acrylamide polymerization. At this temperature range, gelation should be noticeable within 15-30 minutes for typical acrylamide polymerization and within 30-60 minutes for photopolymerization.
Applications Of Polyacrylamide Gel Electrophoresis
1. Separation of proteins
PAGE is considered the technique of choice for the separation of proteins by electrophoresis due to its flexibility in the modification in the setup.
1.1 Protein PAGE is usually conducted in a discontinuous buffer system.
In most cases, electrophoretic separation of proteins is resolved in a discontinuous buffer system, called disc electrophoresis. In this system, protein samples are resolved in a polyacrylamide gel consisting of a lower acrylamide-concentrated stacking gel on the upper layer and a higher acrylamide-concentrated resolving gel. The stacking gel is made from 5% acrylamide gel and contains sample application wells. The migration of the components in the stacking gel is primarily based on their net charge, and the components are “stacked” in the order of their mobility at the border between the stacking and the resolving gels. In the resolving gel, the components encounter frictional force, which causes them to migrate based on both their size and net charge.
1.2 PAGE can resolve proteins in their natural conformation or their denatured state
Proteins can be resolved based on their mobility and molecular weight in their natural conformation using a native PAGE or in their denatured state based solely on their size using an SDS-PAGE. SDS-PAGE is more widely used for the determination of the protein size because the SDS has masked the overall net charge of the proteins and unfolded them, rendering their shape and conformation irrelevant to their mobility during electrophoresis.
Nonetheless, native PAGE is particularly popular for the analysis of membrane proteins and multiprotein complexes. This is because membrane proteins are usually dissolved in detergent-containing buffers that can interfere with the SDS, and the identification of the components in multiprotein complexes is more informative in the light of their functionality, which can be achieved using the native PAGE that takes into account their native conformation.
Both PAGEs can be performed in either continuous and discontinuous buffer systems. Native PAGE and SDS-PAGE can be combined in a two-dimensional gel electrophoresis (2D-PAGE) so that higher resolution and more information are acquired. For example, proteins can be resolved based on their net charge using native PAGE in the first dimension, followed by SDS-PAGE in the second so that the resolved components are further separated based on their mass.
2. Separation of nucleic acids
Unlike proteins, PAGE is not the preferable technique in the separation of nucleic acids due to the toxicity of non-polymerized polyacrylamide gels and the complexity in the preparation, when compared with agarose gels. Nevertheless, the finer porosity of the polyacrylamide gels allows PAGE to complement agarose gel electrophoresis. The followings are examples of the application of PAGE separation of nucleic acids that cannot be performed using agarose gel electrophoresis:
2.1. DNA sequencing
PAGE originally constitutes the last step in DNA sequencing based on Sanger and Maxam-Gilbert methods. In DNA sequencing, several DNA fragments of many lengths, each terminated with different species of nucleobases and differed by one nucleotide, are separated in a denaturing polyacrylamide gel. The pattern of migration of the DNA fragments is analyzed from the bottom to the top to obtain the sequence information. Nowadays, with the development of fluorescent tags and capillary electrophoresis that can automatically record the readouts, DNA sequencing is typically performed using automated sequencing.
2.2. Detection of polymorphisms and mutations
In PAGE, the mobility of nucleic acids is not only influenced by the length of the sequence but also by the nucleobases in the sequence. Changes in the nucleotide sequence will lead to changes in the secondary structure of the double-stranded DNA. Here, the amplified DNA fragment of interest is heated together with that of the wild-type, and the resulting heteroduplexes are separated on a native PAGE to unveil the mobility shift of the heteroduplexes, in comparison to the corresponding homoduplexes and the single-stranded DNA molecules.
Overall, the characteristics of polyacrylamide gels have made PAGE one of the most versatile techniques in gel electrophoresis despite the complicated preparation and the toxicity of the gel components. The adaptability of polyacrylamide allows it to complement agarose and other matrices, which have made PAGE one of the earliest forms of electrophoresis that is essential and still relevant in biotechnology.
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