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Zymography is a technique for detecting, identifying, and quantifying enzyme activity through gel electrophoresis. Gelatin zymography is used to detect matrix metalloproteinase (MMP) activity (a class of zinc-dependent endopeptidases) such as MMP-2 and MMP-9. We describe a semiquantitative method, optimized for determining the activity of secreted gelatinases in cell culture conditioned medium via the digestion of gelatin embedded in a polyacrylamide gel. This assay can also be partnered with a quantitative “invadopodia degradation” assay, which detects the degradation of FITC-labelled gelatin by actin-rich membrane protrusions known as invadopodia. These projections are enriched in proteolytic enzymes, including MMP-2 and MMP-9 in glioblastoma cells, and are secreted to modify/degrade ECM proteins and facilitate tumor cell invasion.

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Gelatin Zymography: Detecting Gelatinase Activity as a Partner for Invadopodia-Mediated Invasion by Tumor Cells

  • 实验方案

  • 发表日期：

    2025年

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  • Marija Dinevska,
  • Samuel Widodo,
  • Chantel McEntee,
  • Theo Mantamadiotis,
  • Stanley S. Stylli

摘要

Zymography is a technique for detecting, identifying, and quantifying enzyme activity through gel electrophoresis. Gelatin zymography is used to detect matrix metalloproteinase (MMP) activity (a class of zinc-dependent endopeptidases) such as MMP-2 and MMP-9. We describe a semiquantitative method, optimized for determining the activity of secreted gelatinases in cell culture conditioned medium via the digestion of gelatin embedded in a polyacrylamide gel. This assay can also be partnered with a quantitative “invadopodia degradation” assay, which detects the degradation of FITC-labelled gelatin by actin-rich membrane protrusions known as invadopodia. These projections are enriched in proteolytic enzymes, including MMP-2 and MMP-9 in glioblastoma cells, and are secreted to modify/degrade ECM proteins and facilitate tumor cell invasion.