Insulin-like growth factor binding protein (IGFBP) substrate zymography. A new tool to identify and characterize IGFBP-degrading proteinases

Endocrine
J L FowlkesH Nagase

Abstract

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 zymogr...Continue Reading

Citations

Sep 1, 1999·Clinical Endocrinology·E NieschlagG F Weinbauer
Apr 25, 2001·Clinical Endocrinology·B JinD J Handelsman
Apr 6, 1999·APMIS : Acta Pathologica, Microbiologica, Et Immunologica Scandinavica·P HenrietY A Declerck
Jul 23, 1999·Annals of the New York Academy of Sciences·J L FowlkesK M Thrailkill
Dec 25, 2010·Integrative Biology : Quantitative Biosciences From Nano to Macro·Miles A MillerDouglas A Lauffenburger

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