What is meant by 'electrophoresis' ? Enumerate the working principle of sds polyacrylamide gel electrophoresis with illustration. State two applications of this technique. Biological discussion
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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.[1]
Hydration of acrylonitrile results in formation of acrylamide molecules (C3H5NO) by nitrile hydratase.[2] 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 water it polymerizes resulting in formation of polyacrylamide.[3] It is useful to make polyacrylamide gel via acrylmide 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. At the pH at which gel electrophoresis is carried out the SDS molecules are negatively charged and bind to proteins in a set ratio, approximately one molecule of SDS for every 2 amino acids.[4] In this way, the detergent provides all proteins with a uniform charge-to-mass ratio. By binding to the proteins the detergent destroys their secondary, tertiary and/or quaternary structure denaturing them and turning them into negatively charged linear polypeptide chains. When subjected to an electric field in PAGE, the negatively charged polypeptide chains travel toward the anode with different mobility. Their mobility, or the distance traveled by molecules, is inversely proportional to the logarithm of their molecular weight.[5] By comparing the relative ratio of the distance traveled by each protein to the length of the gel (Rf) one can make conclusions about the relative molecular weight of the proteins, where the length of the gel is determined by the distance traveled by a small molecule like a tracking dye.[6]
Proteins of the erythrocyte membrane separated by SDS-PAGE according to their molecular masses
SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) is a variant of polyacrylamide gel electrophoresis, an analytical method in biochemistry for the separation of charged molecules in mixtures by their molecular masses in an electric field. It uses sodium dodecyl sulfate (SDS) molecules to help identify and isolate protein molecules.
SDS-PAGE is a discontinuous electrophoretic system developed by Ulrich K. Laemmli which is commonly used as a method to separate proteins with molecular masses between 5 and 250 KDa.[1] The publication describing it is the most frequently cited paper by a single author, and the second most cited overall.