Zymography of Metalloproteinases

Linda Troeberg1, Hideaki Nagase1

1 Imperial College London, London, United Kingdom
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 21.15
DOI:  10.1002/0471140864.ps2115s33
Online Posting Date:  November, 2004
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Zymography is an electrophoretic technique enabling visualization of the number and approximate size of peptidases in a sample on the basis of their hydrolysis of a protein substrate within the gel. The technique is particularly useful for analyzing the peptidase composition of complex biological samples because visualization depends directly on proteolytic activity. This unit presents a representative zymography protocol for the study of matrix metallopeptidases (MMPs).

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Table of Contents

  • Basic Protocol 1: Zymography of Matrix Metalloproteinases Using a Gelatin or Casein Substrate
  • Alternate Protocol 1: Zymography of Matrix Metalloproteinases Using Substrate Proteins other than Gelatin
  • Alternate Protocol 2: Real‐Time Zymography
  • Alternate Protocol 3: Reverse Zymography for Study of Proteinase Inhibitors
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Zymography of Matrix Metalloproteinases Using a Gelatin or Casein Substrate

  • Acrylamide/bisacrylamide solution (see recipe)
  • Separating gel buffer (see recipe)
  • 10× casein or gelatin substrate solution (see recipe)
  • Sucrose solution (see recipe)
  • 10% (w/v) ammonium persulfate (see recipe)
  • n‐Butanol, H 2O saturated
  • Stacking gel buffer (see recipe)
  • MMP sample
  • 1 mM APMA (see recipe)
  • 2× sample loading buffer (see recipe)
  • Proteinase standards (e.g., recombinant MMP‐1, ‐2, or ‐3)
  • 1× anode and cathode reservoir buffers (see recipe)
  • Sample known to degrade gelatin or casein (e.g., recombinant culture supernatents)
  • Molecular mass standards (e.g., BioRad low‐range SDS‐PAGE standards)
  • Enzyme renaturing buffer (see recipe)
  • Developing buffer, 37°C (see recipe)
  • Staining solution (see recipe)
  • Destaining solution (see recipe)
  • Electrophoresis apparatus (e.g., ∼9‐cm × 6‐cm × 0.75‐mm minigel system) and comb
  • Gel‐loading pipet tips or Hamilton syringe
  • Constant voltage power supply
  • Sealed plastic container
  • Additional equipment and reagents for preparing and running acrylamide gels (unit 10.1)
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Literature Cited

Literature Cited
   Barrett, A.J. 1966. Chondromucoprotein‐degrading enzymes. Nature 211:1188‐1190.
  This work is supported by a Wellcome Trust grant no. 057508. The authors thank Abhilash Jain, Nidhi Sofat, and Yoshifumi Itoh for providing conditioned medium from cultured human wrist joint synovium, human chondrocytes, and TPA‐treated HT1080, respectively.
   Barrett, A.J. 1981. Alpha 2‐macroglobulin. Methods Enzymol. 80C:737‐754.
   Bury, A.F. 1981. Analysis of protein and peptide mixtures. Evaluation of three sodium dodecyl sulfate‐polyacrylamide gel electrophoresis buffer systems. J. Chromatogr. 213:491‐500.
   Feitosa, L., Gremski, W., Veiga, S.S., Elias, M.C., Graner E., Mangili, O.C., and Brentani, R.R. 1998. Detection and characterization of metalloproteinases with gelatinolytic, fibronectinolytic and fibrinogenolytic activities in brown spider (Loxosceles intermedia) venom. Toxicon 36:1039‐1051.
   Gogly, B., Groult, N., Hornebeck, W., Godeau, G., and Pellat, B. 1998. Collagen zymography as a sensitive and specific technique for the determination of subpicogram levels of interstitial collagenase. Anal. Biochem. 255:211‐216.
   Hattori, S., Fujisaki, H., Kiriyama, T., Yokoyama, T., and Irie, S. 2002. Real‐time zymography and reverse zymography: A method for detecting activities of matrix metallopeptidases and their inhibitors using FITC‐labeled collagen and casein as substrates. Anal. Biochem. 301:27‐34.
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