Zymography in Multiwells for Quality Assessment of Proteinases.

Methods Mol Biol

Regional Medical Research Centre, Indian Council of Medical Research, Port Blair, India.

Published: March 2018

AI Article Synopsis

  • Zymography is a standardized method for analyzing proteinases but can be challenging for large sample sizes using traditional electrophoresis.
  • A solution involves using substrate-impregnated gels in multiwell formats, allowing for parallel assays of enzymes copolymerized with gelatin substrates.
  • This method, which eliminates the need for electrophoresis and electricity, has been successfully tested for specific collagenases and metalloproteinases and allows for characterizing enzyme activity with different cations and regulatory molecules.

Article Abstract

Zymography is a well-standardized protocol for the qualitative assessment and analysis of proteinases under specified conditions. However, analysis of a large number of samples simultaneously becomes a challenge when the zymography is carried out by the usual protocol of electrophoresis. This can be overcome by assaying the matrix-degrading proteinases in substrate-impregnated gels in multiwells. Enzymes are copolymerized with 300 mL of 10% acrylamide impregnated with gelatin substrate and incubated for 16 h. The gels are then stained with Coomassie blue, destained with water, and visualized with the naked eye. The intensity; if needed can be measured with a densitometer or gel documentation system. This method has been tested for bacterial collagenases as well as some matrix-degrading metalloproteinases that were purified from rat mammary gland. It can also be used to characterize the enzymes with respect to the type and concentration of the cations required for activity and the role of other regulatory molecules that may affect the enzyme activity. The added advantage of this method is that the electrophoresis set up and electricity is not needed for the procedure.

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http://dx.doi.org/10.1007/978-1-4939-7111-4_21DOI Listing

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Article Synopsis
  • Zymography is a standardized method for analyzing proteinases but can be challenging for large sample sizes using traditional electrophoresis.
  • A solution involves using substrate-impregnated gels in multiwell formats, allowing for parallel assays of enzymes copolymerized with gelatin substrates.
  • This method, which eliminates the need for electrophoresis and electricity, has been successfully tested for specific collagenases and metalloproteinases and allows for characterizing enzyme activity with different cations and regulatory molecules.
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