Zymography

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Plasmodium knowlesi hemoglobinase imprint.

The zymography is on the SDS-PAGE -based method in which a substrate was added to the gel for a subject to be examined enzyme or protein mixture.

principle

By adding a substrate to the gel mixture, the activity of an enzyme can be detected after an electrophoretic separation . So z. B. when an amylase is detected, starch is added, while casein or gelatin is used for proteases . In contrast to SDS-PAGE, the proteins in the SDS sample buffer are not boiled, so that no denaturation takes place. Depending on the enzyme, the reducing agents are also omitted. After the electrophoresis, the SDS is extracted with a Triton X-100 solution and the surfactant is exchanged at the same time . Following the SDS extraction, the gel is placed in a buffer suitable for the enzyme , which usually increases the enzymatic activity. After an exposure time, the substrate or the product is colored using suitable detection methods, e.g. B. Lugol's solution for starch or for proteins the Coomassie , Amido black , Ponceau S or silver coloring . In the case of post-translational modifications , the changes can be stained by immunostaining with appropriately specific antibodies . After zymography, the degradation of the substrate or the production of the product is shown in the colored gel, which is referred to as an anti-zymogram or a zymogram .

Individual evidence

  1. ↑ E. Hempelmann, B. Putfarken, K. Rangachari, RJM Wilson: Immunoprecipitation of malarial acid endopeptidase . In: Parasitology . 92, No. 02, 1986, pp. 305-312. doi : 10.1017 / S0031182000064076 . PMID 3520446 .
  2. ↑ MS Lantz, P. Ciborowski: Zymographic techniques for detection and characterization of microbial proteases. . In: Methods Enzymol. . 235, 1994, pp. 563-594. doi : 10.1016 / 0076-6879 (94) 35171-6 . PMID 8057927 .
  3. ↑ TF Martínez, FJ Alarcón, M. Díaz-López, FJ Moyano: Improved detection of amylase activity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with copolymerized starch. . In: Electrophoresis . 21, No. 14, 2000, pp. 2940-2943. doi : 10.1002 / 1522-2683 (20000801) 21:14 <2940 :: AID-ELPS2940> 3.0.CO; 2-S . PMID 11001307 .
  4. ^ PA Snoek-van Beurden, JW Von den Hoff: Zymographic techniques for the analysis of matrix metalloproteinases and their inhibitors . In: Biotechniques . 38, No. 1, 2005, pp. 73-83. doi : 10.2144 / 05381RV01 . PMID 15679089 .