Insulin-Like Growth Factor Binding Protein (IGFBP) Substrate Zymography: A New Tool to Identify and Characterize IGFBP-Degrading Proteinases

John L. Fowlkes, Kathryn M. Thrailkill, Delila M. Serra, Hideaki Nagase

Research output: Contribution to journalArticlepeer-review

9 Scopus citations


Insulin-like growth factor binding protein (IGFBP) degrading proteinase activities have been described in biological fluids and conditioned media from numerous cell lines. To identify and characterize IGFBP-degrading proteinases, our laboratory has developed IGFBP substrate zymography. Herein, we illustrate how IGFBP substrate zymography can be used both to identify candidate IGFBP-degrading proteinases and characterize their degradative capabilities. For this purpose, human matrix metalloproteinase-3 (MMP-3), a proteinase that degrades IGFBP-3 in human fibroblast cultures, was first electrophoresed through a polyacrylamide gel containing IGFBP-3 as substrate and then analyzed for its ability to degrade the substrate into immunoreactive fragments that were absorbed onto a polyvinylidene difluoride membrane. IGFBP-3 substrate zymography was capable of detecting as little as 20 ng of human MMP-3, demonstrating a sensitivity similar to casein substrate zymography. Using the zymogram as a template, MMP-3 was identified in a standard SDS-polyacrylamide gel run in parallel with the zymogram, and the corresponding area of the gel was excised. Electroelution of the gel slice yielded active MMP-3 when examined by casein substrate zymography. Furthermore, digestion of IGFBP-3 in solution by the electroeluted MMP-3 revealed the same fragmentation pattern of the binding protein as that produced by MMP-3, which had not been electro-eluted. Together, these studies demonstrate that IGFBP substrate zymography can be a useful tool for both the identification and the characterization of IGFBP-degrading proteinases.

Original languageEnglish
Pages (from-to)33-36
Number of pages4
Issue number1
StatePublished - 1997


  • Insulin-like growth factor binding proteins
  • Matrix
  • Metalloproteinase
  • Proteinase
  • Zymography

ASJC Scopus subject areas

  • Endocrinology, Diabetes and Metabolism
  • Endocrinology


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