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Related Experiment Video

Updated: Feb 28, 2026

Detection of Protease Activity by Fluorescent Peptide Zymography
09:56

Detection of Protease Activity by Fluorescent Peptide Zymography

Published on: January 20, 2019

13.6K

Transfer Zymography.

Daniel Pan1,2, Karl A Wilson1, Anna Tan-Wilson3

  • 1Department of Biological Sciences, State University of New York at Binghamton, Binghamton, NY, 13902, USA.

Methods in Molecular Biology (Clifton, N.J.)
|June 14, 2017
PubMed
Summary

Transfer zymography improves protease analysis by separating enzymes from substrates during electrophoresis. This novel technique enhances proteomic analysis accuracy and simplifies protease band identification.

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Area of Science:

  • Biochemistry
  • Proteomics
  • Molecular Biology

Background:

  • Conventional zymography faces limitations with protease migration and proteomic analysis due to substrate presence in resolving gels.
  • High background substrate protein interferes with accurate protease identification via LC-MS/MS.

Purpose of the Study:

  • To introduce transfer zymography, a technique overcoming conventional zymography's limitations.
  • To enable accurate protease mass estimation and simplify proteomic analysis.

Main Methods:

  • Proteolytic enzymes are resolved using nonreducing SDS-PAGE without substrate in the resolving gel.
  • Enzymes are transferred to a substrate-containing gel (similar to Western blotting) for activity visualization.
  • Simultaneous electrophoresis on a second gel allows for excision of active protease bands for proteomic analysis.
Keywords:
Electrophoretic transferNonreducing SDS-PAGEProteaseProteolytic enzymeProteomicsZymography

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Related Experiment Videos

Last Updated: Feb 28, 2026

Detection of Protease Activity by Fluorescent Peptide Zymography
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Detection of Functional Matrix Metalloproteinases by Zymography
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Main Results:

  • Transfer zymography prevents anomalous migration and inaccurate mass estimation of proteases.
  • Clear bands of protease activity are visualized against a stained background after SDS removal and incubation.
  • Excised bands from a parallel gel facilitate precise proteomic identification.

Conclusions:

  • Transfer zymography offers a superior method for resolving and analyzing proteolytic enzymes.
  • The technique enhances the accuracy of protease mass determination and streamlines proteomic analysis.