SDSPAGE

SDSPAGE, often written as SDS-PAGE, refers to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The term SDSPAGE is a common misspelling of the standard acronym SDS-PAGE, which is the widely used method in biochemistry and molecular biology for separating proteins by size.

Principle and overview

SDS-PAGE relies on an anionic detergent, sodium dodecyl sulfate, to denature proteins and coat them with a uniform negative charge-to-mass ratio. When samples are loaded into a polyacrylamide gel and subjected to an electric field, smaller proteins migrate faster through the gel matrix than larger ones. The gel matrix is typically divided into a stacking layer and a resolving layer, with the latter controlling separation based on molecular weight.

Procedure outline

Proteins are prepared with SDS and often a reducing agent such as DTT or beta-mercaptoethanol, then heated to unfold the proteins. Samples are loaded with a loading buffer and run in an appropriate buffer system, usually Tris-Glycine-SDS. The polyacrylamide concentration is chosen to optimize resolution for the expected protein sizes. After electrophoresis, the gel is stained to visualize protein bands.

Visualization and analysis

Staining with Coomassie Brilliant Blue, silver stain, or fluorescent dyes reveals bands representing proteins. Band positions are compared to molecular weight standards to estimate sizes. Densitometry can provide semi-quantitative assessment of abundance.

Applications and limitations

SDS-PAGE is used to assess protein purity, estimate molecular weight, monitor purification steps, and precede Western blotting. It denatures proteins, so native structure and activity are not preserved. The method requires careful handling of acrylamide and SDS.

History

The technique was popularized in the 1970s by Laemmli, becoming a foundational tool in protein analysis.