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Arpa Sutipatanasomboon is a research scientist based in the Bangkok Metropolitan Region. She began her scientific journey at Mahidol University in Bangkok, Thailand, where she obtained her Bachelor’s in Plant Sciences. Following her passion for research, Arpa moved to Germany to pursue her graduate studies at the University of Cologne. She subsequently earned her Master’s in Biological Sciences and completed her Ph.D. working on the intersection between cell death and proteostasis in Arabidopsis thaliana. Apart from research, she has developed interests in technology transfer, intellectual property, and IP management. Her goal is to use her research and expertise to assist plant breeders and to make science and scientific knowledge accessible to everyone.
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Arpa Sutipatanasomboon is a research scientist based in the Bangkok Metropolitan Region. She began her scientific journey at Mahidol University in Bangkok, Thailand, where she obtained her Bachelor’s in Plant Sciences. Following her passion for research, Arpa moved to Germany to pursue her graduate studies at the University of Cologne. She subsequently earned her Master’s in Biological Sciences and completed her Ph.D. working on the intersection between cell death and proteostasis in Arabidopsis thaliana. Apart from research, she has developed interests in technology transfer, intellectual property, and IP management. Her goal is to use her research and expertise to assist plant breeders and to make science and scientific knowledge accessible to everyone.
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Latest Posts
  • Enzymology
  • SDS-Polyacrylamide Gel Electrophoresis at Neutral pH (NuPAGE)
  • SDS-Polyacrylamide Gel Electrophoresis at Neutral pH (NuPAGE)

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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.[1,2] 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.[1,3]

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.[4,5]

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.[6,7] The polymerization is catalyzed by ammonium persulfate or potassium persulfate and by N,N,N’,N’-tetramethylethylenediamine (TEMED).[6] 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.[3,5] Bis-acrylamide is cross-linked between acrylamide molecules in the polymerizing chain, forming a transparent, charge-neutral and inert polyacrylamide gel.[3,6,7]

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.[5]  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.[5,7]

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.[3,5]

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.[3,5,7] 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: