When an electric current is applied across the gel,the negatively-charged proteins migrate across the gel to the positive pole. The proteins denatured by SDS are applied to one end of a layer of polyacrylamide gel submerged in a buffer.Buffer provide uniform pH and ions for conducting electric potential. Two SDS-PAGE-gels after a completed run Electrophoresis The samples are treated with SDS (sodium dodecyl sulfate),an anionic detergent which denatures the protein by forming complexes with the protein and introducing negative charge to each protein in proportion to its mass.Without SDS,different proteins with similar molecular weights would migrate differently due to differences in folding,as differences in folding patterns would cause some proteins to better fit through the gel matrix than others.SDS linearizes the proteins so that they may be separated strictly by molecular weight.The SDS binds to the protein in a ratio of approximately 1.4 g SDS per 1.0 g protein ],giving an approximately uniform mass:charge ratio for most proteins,so that the distance of migration through the gel can be assumed to be directly related to only the size of the protein.Proteins may be further treated with reducing agent, such as dithiothreitol(DTT) or TRP(Tributyl phosphine)to break any disulfide bonds and then alkalated with iodoacetamide to prevent reformation of disulfide bonds.Ī tracking dye like bromophenol blue may be added to the protein solution to track the progress of the protein solution through the gel during the electrophoretic run. Standard gels are typically composed of two layers, one top-most layer called the stacking gel and a lower layer called separating or resolving gel.The stacking layer contains a low percentage of acylamide and has low pH, while the acrylamide concentration of the separating gel varies according to the samples to be run and has higher pH.The difference in pH and acrylamide concentration at the stacking and separating gel provides better resolution and sharper bands in the separating gel. The gel used for SDS-PAGE is made out of acrylamide which form cross-linked polymers of polyacrylamide.

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