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Concept
Polyacrylamide gel electrophoresis
Summary
Polyacrylamide gel electrophoresis (PAGE) is a technique widely used in biochemistry, forensic chemistry, genetics, molecular biology and biotechnology to separate biological macromolecules, usually proteins or nucleic acids, according to their electrophoretic mobility. Electrophoretic mobility is a function of the length, conformation, and charge of the molecule. Polyacrylamide gel electrophoresis is a powerful tool used to analyze RNA samples. When polyacrylamide gel is denatured after electrophoresis, it provides information on the sample composition of the RNA species.
Hydration of acrylonitrile results in formation of acrylamide molecules () by nitrile hydratase. Acrylamide monomer is in a powder state before addition of water. Acrylamide is toxic to the human nervous system, therefore all safety measures must be followed when working with it. Acrylamide is soluble in water and upon addition of free-radical initiators it polymerizes resulting in formation of polyacrylamide. It is useful to make polyacrylamide gel via acrylamide hydration because pore size can be regulated. Increased concentrations of acrylamide result in decreased pore size after polymerization. Polyacrylamide gel with small pores helps to examine smaller molecules better since the small molecules can enter the pores and travel through the gel while large molecules get trapped at the pore openings.
As with all forms of gel electrophoresis, molecules may be run in their native state, preserving the molecules' higher-order structure. This method is called native-PAGE. Alternatively, a chemical denaturant may be added to remove this structure and turn the molecule into an unstructured molecule whose mobility depends only on its length (because the protein-SDS complexes all have a similar mass-to-charge ratio). This procedure is called SDS-PAGE. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a method of separating molecules based on the difference of their molecular weight.
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