Polyacrylamide gel electrophoresis

Polyacrylamide gel electrophoresis (PAGE), describes a technique widely used in biochemistry, forensics, genetics, molecular biology and biotechnology to separate biological macromolecules, usually proteins or nucleic acids, according to their electrophoretic mobility. Mobility is a function of the length, conformation and charge of the molecule.

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 and mass-to-charge ratio. This procedure is called SDS-PAGE. For nucleic acids, urea is the most commonly used denaturant. For proteins, sodium dodecyl sulfate (SDS) is an anionic detergent applied to protein samples to coat proteins in order to impart two negative charges for every molecule to every two amino acids of the denatured protein. 2-Mercaptoethanol may also be used to disrupt the disulfide bonds found between the protein complexes, which helps further denature the protein. In most proteins, the binding of SDS to the polypeptide chain imparts an even distribution of charge per unit mass, thereby resulting in a fractionation by approximate size during electrophoresis. Proteins that have a greater hydrophobic content, for instance, many membrane proteins, and those that interact with surfactants in their native environment, are intrinsically harder to treat accurately using this method, due to the greater variability in the ratio of bound SDS. Procedurally, using both Native and SDS Page together, can be used to purify and to separate the various subunits of the protein. Native Page keeps the oligomeric form intact and will show a band on the gel that is representative of the level of activity. Whereas, SDS Page will denature and separate the oligomeric form into its monomers and will show bands that is representative to their molecular weights. These bands can be used to access the purity of and identify the protein.

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