Zymography

Zymography is a method based on SDS -PAGE, wherein a substrate for an enzyme to be tested or protein mixture was added to the gel.

Principle

By adding a substrate in the gel mixture can be detected after electrophoretic separation, the activity of an enzyme. It is added for example in the detection of amylase starch while using in proteases casein or gelatin. In contrast to the SDS- PAGE, the proteins in the SDS- sample buffer is not boiled, so that no denaturation occurs, depending on the enzyme and the reducing agent can be omitted. After electrophoresis, the SDS is extracted with a Triton X-100 solution, and simultaneously replace the surfactant. Following the extraction, the SDS- gel is placed in an appropriate buffer for the enzyme, which is usually increased enzymatic activity. After an exposure, the substrate or the product will be stained with appropriate detection methods, such as Lugol's solution for starch or proteins in the Coomassie -, amido black, Ponceau S or silver staining. In post-translational modifications, the modifications can be stained by immunostaining with corresponding specific antibodies. By zymography in the stained gel shows the degradation of the substrate or the production of the product, which is referred to as an anti- zymogram or as zymogram.