SDS-PAGE

SDS-PAGE stands for Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis. It is a technique used in biochemistry and molecular biology to separate proteins according to their size (length of polypeptide chain).

Procedure

The solution of proteins to be analyzed is first mixed with SDS, an anionic detergent which denatures the protein's secondary and non–disulfide–linked tertiary structures, and applies a negative charge to every protein. 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. Adding SDS solves this problem, as it linearizes the proteins so that they may be separated strictly by length (primary structure, or number of amino acids). The SDS binds to the protein in a ratio of approximately 1.4 g SDS per 1.0 g protein (although binding ratios can vary from 1.1-2.2 g SDS/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. A tracking dye may be added to the protein solution to allow the experimentor to track the progress of the protein solution through the gel during the electrophoretic run.

Besides the addition of SDS, proteins may optionally be boiled in the presence of a reducing agent, such as dithiothreitol (DTT) or 2-mercaptoethanol, which further denatures the proteins by reducing disulfide linkages, thus overcoming some forms of tertiary protein folding, and breaking up quaternary protein structure (oligomeric subunits). This is known as reducing SDS-PAGE, and is most commonly used. Non-reducing SDS-PAGE (no boiling and no reducing agent) may be used when native structure is important in further analysis (e.g. enzyme activity, shown by the use of zymograms).

The denatured proteins are subsequently applied to one end of a layer of polyacrylamide gel submerged in a suitable buffer. An electric current is applied across the gel, causing the negatively-charged proteins to migrate across the gel. Depending on their size, each protein will move differently through the gel matrix: short proteins will more easily fit through the pores in the gel, while larger ones will have more difficulty. After a set amount of time (usually a few hours), the proteins will have differentially migrated based on their size; smaller proteins will have traveled farther down the gel, while larger ones will have remained closer to the point of origin. Thus proteins may be separated roughly according to size (and therefore, molecular weight). Following electrophoresis, the gel may be stained (most commonly with Coomassie Brilliant Blue or silver stain), allowing visualisation of the separated proteins, or processed further (e.g. Western blot). After staining, different proteins will appear as distinct bands within the gel. It is common to run "marker proteins" of known molecular weight in a separate lane in the gel, in order to calibrate the gel and determine the weight of unknown proteins by comparing the distance traveled relative to the marker.

Gel electrophoresis is usually the first choice as an assay of protein purity due to its reliability and ease. The presence of SDS and the denaturing step causes proteins to be separated solely based on size. False negatives and positives are possible. A comigrating contaminant can appear as the same band as the desired protein. This comigration could also cause a protein to run at a different position or to not be able to penetrate the gel. This is why it is important to stain the entire gel including the stacking section. Coomassie blue will also bind with less affinity to glycoproteins and fibrous proteins, which interferes with quantitation (Deutcher 1990).

See also

• Western blot

References

• Deutscher MP (ed.) (1990). Guide to protein purification. Methods Enzymol. Vol 182. Academic Press Inc. San Diego, CA.
• Sørensen BK, Højrup P, Østergård E, Jørgensen CS, Enghild J, Ryder LR, Houen G (2002). Silver staining of proteins on electroblotting membranes and intensification of silver staining of proteins separated by polyacrylamide gel electrophoresis. Anal Biochem 304: 33-41.
• Schägger H, von Jagow G (1987). Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166: 368-379.
• Laemmli UK (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685.